Method For Generating Aptamers with Improved Off-Rates

ABSTRACT

The present disclosure describes methods for producing aptamers and photoaptamers having slower dissociation rate constants than are obtained using prior SELEX and photoSELEX methods. The disclosure further describes aptamers and photoaptamers having slower dissociation rate constants than those obtained using prior methods. This invention relates to the field of diagnostic histology, cytology, histopathology, and cytopathology methods and reagents for the detection of various disease states. More specifically, the invention relates to the use of aptamers in histologic, cytologic, histopathic, and/or cytopathic diagnostic methods. Aptamers may be provided that react with specific target molecules contained within a histological or cytological sample. Aptamers may be used to assess localization, relative density, and presence or absence of one or more target. Targets may be selected that are specific and diagnostic of a given disease state for which the sample was collected. Aptamers may be used to introduce target specific signal moieties. Antigen retrieval methods may be applied to the sample prior to reaction with the specific aptamer/s to improve interaction of the aptamer and target within the sample. Or aptamers may be developed for the specific target that eliminates the need for the antigen retrieval process. In addition to target identification, aptamers may be used to amplify signal generation through a variety of methods.

RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.12/175,434, filed Jul. 17, 2008, which claims the benefit of U.S.Provisional Application Ser. No. 60/950,281, filed Jul. 17, 2007, U.S.Provisional Application Ser. No. 60/950,293, filed Jul. 17, 2007, U.S.Provisional Application Ser. No. 60/950,283, filed Jul. 17, 2007, U.S.Provisional Application Ser. No. 61/031,420, filed Feb. 26, 2008 andU.S. Provisional Application Ser. No. 61/051,594, filed May 8, 2008.This application is also a continuation in part of U.S. application Ser.No. 11/623,580 and U.S. application Ser. No. 11/623,535, each of whichwas filed on Jan. 16, 2007. Each of these references is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates generally to methods for the generationof aptamers and photoaptamers having improved properties and theimproved aptamers and photoaptamers generated thereby. In particular,the present disclosure describes slow off-rate aptamers that are highlyspecific to a target of interest. The disclosure describes thecomposition of these slow off-rate aptamers as well methods for theirselection. Further the disclosure describes aptamer constructs withimproved functionalities for detection methods. Further, the disclosuredescribes applications enabled by these improved aptamers such asmethods for improving identification of one or more specific markers inhistological and/or cytological specimens using marker specific aptamersfor the diagnosis of a disease state.

BACKGROUND

The following description provides a summary of information relevant tothe present disclosure and is not a concession that any of theinformation provided or publications referenced herein is prior art tothe claimed invention.

The SELEX process is a method for the in vitro selection of nucleic acidmolecules that are able to bind with high specificity to targetmolecules and is described in U.S. Pat. No. 5,475,096 entitled “NucleicAcid Ligands” and U.S. Pat. No. 5,270,163 (see also WO 91/19813)entitled “Nucleic Acid Ligands” each of which is specificallyincorporated by reference herein. These patents, collectively referredto herein as the SELEX patents, describe methods for making an aptamerto any desired target molecule.

The basic SELEX process has been modified to achieve a number ofspecific objectives. For example, U.S. Pat. No. 5,707,796, entitled“Method for Selecting Nucleic Acids on the Basis of Structure” describesthe use of the SELEX process in conjunction with gel electrophoresis toselect nucleic acid molecules with specific structural characteristics,such as bent DNA. U.S. Pat. No. 5,580,737, entitled “High-AffinityNucleic Acid Ligands That Discriminate Between Theophylline andCaffeine” describes a method for identifying highly specific aptamersable to discriminate between closely related molecules, termedCounter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Solution SELEX” describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. Pat. No. 5,496,938, entitled “Nucleic Acid Ligands to HIV-RT andHIV-1 Rev” describes methods for obtaining improved aptamers after SELEXhas been performed. U.S. Pat. No. 5,705,337, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Chemi-SELEX” describesmethods for covalently linking an aptamer to its target. U.S. Pat. No.6,376,424, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Tissue SELEX” describes methods to produce aptamers to cellor tissue specific markers without the purification of the specificmarker.

The SELEX process encompasses the identification of high-affinityaptamers containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides” thatdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′—NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe).

Further modifications of the SELEX process are described in U.S. Pat.No. 5,763,177, U.S. Pat. No. 6,001,577, and U.S. Pat. No. 6,291,184,each of which is entitled “Systematic Evolution of Nucleic Acid Ligandsby Exponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX”; see also, e.g., U.S. Pat. No. 6,458,539, entitled“Photoselection of Nucleic Acid Ligands”. These patents, collectivelyreferred to herein as “the PhotoSELEX patents” describe various SELEXmethods for selecting aptamers containing photoreactive functionalgroups capable of binding and/or photocrosslinking to and/orphotoinactivating a target molecule. The resulting photoreactiveaptamers are referred to as photocrosslinking aptamers or photoaptamers.

Although these SELEX and photoSELEX processes are useful, there isalways a need for processes that lead to improved properties of aptamersgenerated from in vitro selection techniques. For example, a need existsfor aptamers to target molecules with better binding affinities thanthose achieved with naturally occurring DNA or RNA nucleotides, as wellas methods for producing such aptamers. For many applications, such asfor example, in vitro assays, diagnostics, therapeutic, or imagingapplications, it is of interest to produce aptamers with slowdissociation rates from the aptamer/target affinity complex. Severaltechniques have been proposed for producing such reagents (see, e.g., WO99/27133 and US 2005/0003362). However, these selection processes do notdiscriminate between the selection of reagents that have fastassociation kinetics with the target (i.e., fast on-rates) and theselection of reagents that have slow dissociation kinetics with thetarget (i.e., slow off-rates). Thus, there is a need for novel processesand techniques that favor the selection of slow off-rate aptamers whileinhibiting the selection of aptamers that simply have a fast associationrate with the target.

Finally, there is a need for aptamer constructs that include differentbuilt-in functionalities. These functionalities may include tags forimmobilization, labels for detection, means to promote or controlseparation, etc.

Cytology consists of the evaluation of cell morphology, structure, andsub-structure, and as a diagnostic tool can be applied to any bodilyfluid or organ. Specimens may be cells released in a fluid such asurine, gastric, sputum, pleural, spinal fluid, effusions, etc. or may becollected by needle biopsy or aspiration, scraping, or cytologicalbrush. Aspiration biopsy may be performed on the lymph nodes, thyroid,salivary glands, breast, endometrial, or prostate. Cytologicalevaluations are used to evaluate organelle pathology, cell death(necrosis, apoptosis), cellular injury and response, cell aging,amyloidosis, autoimmune diseases, and to discriminate cancer from otherdisease states.

Histology consists of the evaluation of tissue morphology and structurefor the diagnosis of a disease state, with the identification ofmalignancy being largely based on histological information. There arefour major tissue categories in the body-epithelial, connective, muscle,and nervous. Direct microscopic visualization of tissue features isdifficult due to the thickness of the tissue sample. Thereforetechniques have been developed to allow the production of thin,representative sections of the tissue sample for subsequent analysis.

Both cytology and histology samples have been evaluated withimmunological reagents. Antibodies to specific markers have been used tointroduce dyes or other signaling moieties for visualization.Frequently, the immunological methods are more harsh than the standardmethods because of the additional requirement to make the fixed samplepermeable to the immunological reagents so the fixation method may becarefully balanced with the subsequent immunostaining to prevent thegeneration of artifacts. The size of the antibody limits diffusion intofixed cells and tissues. The F_(c) portion of the antibody maynon-specifically associate with cell or tissue structures to generateerroneous results.

Cytologists and histologist are currently being asked to increase thenumber and range of tests conducted on a single collected specimen. Toaccomplish these goals it would be advantageous to have reagents thatcould provide one or more of the following characteristics: (1) beapplied sequentially to the same sample without significant sampledamage; (2) be pre-labeled to reduce or eliminate multiple processsteps; (3) be pre-labeled with a number of different dyes or detectablemoieties that can simultaneously be detected for detection of multipletargets from a single section; (4) eliminate the need for the antigenretrieval process as described below; (5) reduce or eliminate thepermeabilization process; (6) reduce or eliminate non-specificassociation with the non-target; (7) and stabilize label location. Slowoff-rate aptamers could address any of these needs in addition toproviding (1) a more consistent and reliable reagent because they arechemically synthesized; (2) chemically robust reagents that have reducedstorage requirements; and (3) rapid and high-throughput discovery ofbinding reagents to target new proteins.

SUMMARY

The present disclosure describes novel aptamers, and methods to produceand use such aptamers. In particular, the disclosure describes slowoff-rate (slow rate of dissociation) aptamers, slow off-rate aptamerscontaining C-5 modified pyrimidines, and processes for the selection ofslow off-rate aptamers by dilution, by the addition of a competitor, orby a combination of both approaches. In addition, slow off-rate aptamersto various targets such as proteins and peptides are described. Slowoff-rate aptamers with unique structural features and meltingtemperatures are also described. The disclosure also describes slowoff-rate aptamers with photoreactive functional groups, aptamers thatare refractory to the presence of poly-anionic materials, and aselection process for these aptamers, as well as aptamers constructedwith a variety of other functionalities to improve their utility invarious applications.

The present disclosure describes improved SELEX methods for generatingaptamers that are capable of binding to target molecules. Morespecifically, the present disclosure describes methods for producingaptamers and/or photoaptamers having slower rates of dissociation fromtheir respective target molecules than aptamers and photoaptamersobtained with previous SELEX methods. Generally, after contacting thecandidate mixture with the target molecule and allowing the formation ofnucleic acid-target complexes to occur, a slow off-rate enrichmentprocess is introduced wherein nucleic acid-target complexes with fastdissociation rates will dissociate and not reform, while complexes withslow dissociation rates will remain intact. Methods for introducing aslow off-rate enrichment process include, but are not limited to, addingcompetitor molecules to the mixture of nucleic acids and targetmolecules, diluting the mixture of nucleic acids and target molecules,or a combination of both of these. The disclosure further describesaptamers and photoaptamers obtained using these methods.

In one embodiment, the method comprises preparing a candidate mixture ofnucleic acids; contacting the candidate mixture with a target moleculewherein nucleic acids with the highest relative affinities to the targetmolecule preferentially bind the target molecule, forming nucleicacid-target molecule complexes; introducing a slow off-rate enrichmentprocess to induce the dissociation of nucleic acid-target moleculecomplexes with relatively fast dissociation rates; partitioning theremaining bound nucleic acid-target molecule complexes from free nucleicacids in the candidate mixture; and identifying the nucleic acids thatwere bound to the target molecule. The process may further include theiterative step of amplifying the nucleic acids that bind to the targetmolecule to yield a mixture of nucleic acids enriched with nucleic acidsthat bind to the target molecule yet produce nucleic acid-targetmolecule complexes having slow dissociation rates.

In another embodiment, the candidate mixture of nucleic acids includesnucleic acids containing modified nucleotide bases that may aid in theformation of modified nucleic acid-target complexes having slowdissociation rates. Improved methods for performing SELEX with modifiednucleotides, including nucleotides which contain photoactive or otherfunctional groups, or nucleotides which contain placeholders forphotoactive groups are disclosed in U.S. application Ser. No.12/175,388, filed Jul. 17, 2008, entitled “Improved SELEX andPHOTOSELEX,” which is incorporated by reference herein in its entirety.Placeholder nucleotides may also be used for the mid-SELEX or post-SELEXintroduction of modified nucleotides that are not photoreactive.

The various methods and steps described herein can be used to generatean aptamer capable of either (1) binding to a target molecule or (2)binding to a target molecule and subsequently forming a covalent linkagewith the target molecule upon irradiation with light in the UV orvisible spectrum.

In another aspect, the various methods and steps described herein can beused to generate an aptamer capable of modifying the bioactivity of atarget molecule through binding and/or crosslinking to the targetmolecule. In one embodiment, an aptamer to a unique target moleculeassociated with or relevant to a specific disease process is identified.This aptamer can be used as a diagnostic reagent, either in vitro or invivo. In another embodiment, an aptamer to a target molecule associatedwith a disease state may be administered to an individual and used totreat the disease in vivo. The aptamers and photoaptamers identifiedherein can be used in any diagnostic, imaging, high throughput screeningor target validation techniques or procedures or assays for whichaptamers, oligonucleotides, antibodies and ligands, without limitationcan be used. For example, aptamers and photoaptamers identified hereincan be used according to the methods described in detail in U.S.application Ser. No. 12/175,446, filed Jul. 17, 2008, entitled“Multiplexed Analyses of Test Samples”, which is incorporated byreference herein in its entirety.

Various embodiments describe the utility of slow off-rate aptamers forthe identification and visualization of specific targets in cytologicaland/or histological samples and as histology/cytology reagents. In oneembodiment, slow off-rate aptamers are selected for one or more targetsfrom an optionally fixed cellular or tissue sample for use in thatspecific cell or tissue in a diagnostic application. Thus, the slowoff-rate aptamers would specifically recognize the target in the fixedconfiguration (whether that is cross linked or otherwise modified) andin the cellular/tissue environment specific to the diagnosis to be made.Thus, no antigen retrieval process would then be required.

In another embodiment, slow off-rate aptamers are produced withphotoreactive or chemically reactive moieties that can be used tocrosslink the slow off-rate aptamer (or aptamers) to its (their)specific target (targets) within in the cytological or histologicalsample. The ability to make a covalent linkage between the slow off-rateaptamer and the specific target may facilitate the retention of a targetspecific detectable moiety through the sample processing steps requiredwith a histological or cytological preparation.

Other embodiments rely on slow off-rate aptamers that are substantiallysmaller than antibodies and should have improved dispersion capabilitiesin the cell or tissue sample. Still other embodiments rely on theelimination of multiple process steps that could reduce the damage doneto cellular and tissue samples.

Yet more embodiments, involve analyzing multiple targets, with thecorresponding specific slow off-rate aptamers, from a single slide thatwould reduce waste, processing time, time to results, and conserveoriginal specimen for any subsequent testing needs or archival purposes.In some embodiments, these multiple slow off-rate aptamers are reactedwith their corresponding target in a sequential manner. In otherembodiments, these multiple slow off-rate aptamers are reacted withtheir corresponding target in a simultaneous manner. In one embodiment,the one or more slow off-rate aptamers are each produced with adifferent detectable moiety.

In another embodiment, the presence of one or more target(s) identifiedin a histological or cytological sample by the detection of a targetspecific slow off-rate aptamer may be used for the differentiation oftype and origin of a suspected tumor. These targets may include tumorspecific markers as well as tissue specific markers, like hormones. Inanother embodiment, the presence of one or more target(s) identified ina histological or cytological sample by detection of a target specificslow off-rate aptamer may be used for the selection of appropriatetherapeutic agents and to evaluate potential outcome.

In one embodiment, one or more slow off-rate aptamers are used in acytological evaluation of a cell sample and may include one or more ofthe following steps: collecting a cell sample, fixing the cell sample,dehydrating, clearing, immobilizing the cell sample on a microscopeslide, permeabilizing the cell sample, treating for antigen retrieval,staining, destaining, washing, blocking, and reacting with one or moreslow off-rate aptamers in a buffered solution. In another embodiment,the cell sample is produced from a cell block.

In one embodiment, one or more slow off-rate aptamers are used in ahistological evaluation of a tissue sample and may include one or moreof the following steps: collecting a tissue specimen, fixing the tissuesample, dehydrating, clearing, immobilizing the tissue sample on amicroscope slide, permeabilizing the tissue sample, treating for antigenretrieval, staining, destaining, washing, blocking, rehydrating, andreacting with one or more slow off-rate aptamers in a buffered solution.In another embodiment, the fixing and dehydrating steps are replacedwith a freezing step.

In another embodiment, the one or more slow off-rate aptamers reactedwith the histological or cytological sample may serve as the nucleicacid target in a nucleic acid amplification method. The nucleic acidamplification method may include PCR, q-beta replicase, rolling circleamplification, strand displacement, helicase dependent amplification,loop mediated isothermal amplification, ligase chain reaction, andrestriction and circularization aided rolling circle amplification.

In one embodiment, the one or more slow off-rate aptamers are reactedwith the histological or cytological sample after the staining step forthe visualization of morphological and cellular structures. In anotherembodiment, the one or more slow off-rate aptamers are reacted with thehistological or cytological sample before the staining step.

In one embodiment, the one or more slow off-rate aptamers for use in thehistological or cytological evaluation are mixed in a buffered solutionthat may further comprise blocking materials, competitors, detergents,stabilizers, carrier nucleic acid, polyanionic materials, etc.

One embodiment describes a method for the diagnosis of a specificdisease state wherein multiple tissue sections are used. One section isutilized as a negative control. In the procedure for the negativecontrol, the reaction step with one or more slow off-rate aptamers in abuffered solution is replaced with a step reacting only the bufferedsolution in the absence of any slow off-rate aptamer. Another section isstained for the morphological or cellular and subcellular analysisappropriate to the tissue type and disease state. One more section isreacted with one or more slow off-rate aptamer in a buffered solution.

In one embodiment, the one or more slow off-rate aptamers are producedwith a detectable moiety and may be directly detected after reactionwith their respective target or targets following an optional wash stepto remove unreacted slow off-rate aptamer or slow off-rate aptamers. Inother embodiments, the one or more slow off-rate aptamers interactionwith their respective target or targets is detected after the twocomponents of an element to support signal generation are reacted.

In another embodiment, the slow off-rate aptamers are selected,identified, produced, and/or synthesized using an optionally fixedtissue specimen. Prior to the SELEX process, the fixed tissue may betreated to permeabilize sample membranes by methods equivalent to thoseused in the staining process. The method of fixation used in the SELEXprocess may be the same as the fixation method used on a tissue or cellsample in the actual histological/cytological procedure. The requirementis that the target that the slow off-rate aptamer is specific to bepresented to the aptamer library in the selection process in the fixedconfiguration that will be present in the sample to be analyzed.

In another embodiment, the targets detected in the histological orcytological sample by the presence of its cognate slow off-rate aptamermay be used to guide the design of the therapeutic regimen or to predictthe potential response to the therapeutic regimen available.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a number of different stains for histological slides,the material that is stained and the expected color of the stainedmaterial.

FIG. 2 illustrates an exemplary SELEX method which includes the step ofincorporating a slow off-rate enrichment process.

FIG. 3 illustrates representative aptamer template, primer, andcomplementary oligonucleotide sequences used in the disclosure. Theoligonucleotides were prepared by standard solid-phase synthesistechniques. B=dT-biotin.

FIG. 4 illustrates histograms of dissociation rate constants foraffinity aptamers selected without (FIG. 4A) and with (FIG. 4B) a slowoff-rate enrichment process as described in Example 2.

FIGS. 5A and 5B show oligonucleotides that were used to prepare thecandidate mixtures or perform various steps in the selection processdescribed in Example 2. The oligonucleotides were prepared by standardsolid-phase synthesis techniques. BrdU (5-bromo-dUTP), anthraquinone(AQ), and psoralen (Psor) chromophores were purchased asphosphoramidites and added to the 5′ terminus of the forward primerduring synthesis. 4-Azido-2-nitro-aniline (ANA) was prepared as apara-nitro-phenyl carbonate derivative and coupled to a 5′ hexylaminephosphoramidite after synthesis. Two candidate mixture sequences wereused in this example, designated 1 and 2. B=dT-biotin. Template 1 (FIG.5A) was only used with candidate mixtures containing 5′-BrdU, AQ, andANA, and Template 2 (FIG. 5B) was only used with candidate mixturescontaining 5′-Psor for Example 2.

FIG. 6 illustrates the chemical structures of the chromophores coupledto the 5′ terminus of the forward primer as illustrated in FIGS. 5A and5B.

FIG. 7 illustrates a PAGE analysis of crosslink activity of TIMP-35′ANA/BzdU enriched library using 5′-Fixed PhotoSELEX as described inExample 3. The gel illustrates the separation of free aptamer (A_(f)),intramolecular crosslinked aptamer (A_(f)*), and crosslinkedprotein:aptamer complexes (P:A).

FIG. 8 is a chart of over 500 targets for which aptamers have beenidentified. Many of these aptamers have been designed to have slowdissociation rates from their respective targets.

FIGS. 9A to 9D illustrate aptamer constructs that contain a variety ofdifferent and optional functionalities including immobilization tags,labels, photocrosslinking moieties, spacers, and releasable moieties.

FIGS. 10 A to 10F illustrate examples of aptamer constructs including acleavable or releasable element, a tag (for example biotin), a spacer,and a label (for example Cy3).

FIG. 11 illustrates the aptamer and primer constructs described in thedisclosure. Cy3 represents a Cyanine 3 dye, PC a photocleavable linker,ANA a photoreactive crosslinking group, (AB)₂ a pair of biotin residuesseparated by dA residues, and (T)₈ a poly dT linker. Primer constructsare complementary to the complete 3′ fixed region of the aptamerconstructs.

FIGS. 12 A to 12 C illustrate dose response curves for slow off-rateaptamers versus traditional aptamers for three different targets.

FIGS. 13A and 13B illustrate performance curves for a slow off-rateaptamer where the target was a peptide.

FIG. 14 describes the base modifications of nucleotides included in thisdisclosure. The R groups that may be used are described in addition tothe linkers (X) that may be used between the nucleotide attachment pointand the R group. The positions of attachment for the various “R” groupsare also indicated on the respective R group.

FIG. 15 illustrates a plot used in the determination of the bindingconstant for a slow off-rate aptamer containing C-5 modified pyrimidinesto thrombin.

DETAILED DESCRIPTION

The practice of the invention disclosed herein employs, unless otherwiseindicated, conventional methods of chemistry, microbiology, molecularbiology, and recombinant DNA techniques within the level of skill in theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (CurrentEdition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover,ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition; Histology for Pathologists (S.E. Mills, Current Edition). Allpublications, published patent documents, and patent applications citedin this specification are indicative of the level of skill in the art(s)to which the invention pertains. All publications, published patentdocuments, and patent applications cited herein are hereby incorporatedby reference to the same extent as though each individual publication,published patent document, or patent application was specifically andindividually indicated as being incorporated by reference.

As used in this specification, including the appended claims, thesingular forms “a,” “an,” and “the” include plural references, unlessthe content clearly dictates otherwise, and are used interchangeablywith “at least one” and “one or more.” Thus, reference to “an aptamer”includes mixtures of aptamers, reference to “a probe” includes mixturesof probes, and the like.

As used herein, the term “about” represents an insignificantmodification or variation of the numerical values such that the basicfunction of the item to which the numerical value relates is unchanged.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

As used herein, “nucleic acid ligand” “aptamer” and “clone” are usedinterchangeably to refer to a non-naturally occurring nucleic acid thathas or may have a desirable action on a target molecule. A desirableaction includes, but is not limited to, binding of the target,catalytically changing the target, reacting with the target in a waythat modifies or alters the target or the functional activity of thetarget, covalently attaching to the target (as in a suicide inhibitor),and facilitating the reaction between the target and another molecule.In one embodiment, the action is specific binding affinity for a targetmolecule, such target molecule being a three dimensional chemicalstructure, other than a polynucleotide, that binds to the aptamerthrough a mechanism which is predominantly independent of Watson/Crickbase pairing or triple helix binding, wherein the aptamer is not anucleic acid having the known physiological function of being bound bythe target molecule. Aptamers include nucleic acids that are identifiedfrom a candidate mixture of nucleic acids, the aptamer being a ligand ofa given target, by the method comprising: (a) contacting the candidatemixture with the target, wherein nucleic acids having an increasedaffinity to the target relative to other nucleic acids in the candidatemixture may be partitioned from the remainder of the candidate mixture;(b) partitioning the increased affinity and/or slow off-rate nucleicacids from the remainder of the candidate mixture; and (c) amplifyingthe increased affinity, slow off-rate nucleic acids to yield aligand-enriched mixture of nucleic acids, whereby aptamers to the targetmolecule are identified. It is recognized that affinity interactions area matter of degree; however, in this context, the “specific bindingaffinity” of an aptamer for its target means that the aptamer binds toits target generally with a much higher degree of affinity than it bindsto other, non-target, components in a mixture or sample. An “aptamer” or“nucleic acid ligand” is a set of copies of one type or species ofnucleic acid molecule that has a particular nucleotide sequence. Anaptamer can include any suitable number of nucleotides. “Aptamers” referto more than one such set of molecules. Different aptamers may haveeither the same number or a different number of nucleotides. Aptamersmay be DNA or RNA and may be single stranded, double stranded, orcontain double stranded regions.

As used herein, “slow off-rate” or “slow rate of dissociation” or “slowdissociation rate” refers to the time it takes for an aptamers/targetcomplex to begin to dissociate. This can be expressed as a half life,t_(1/2), or the point at which 50% of the aptamer/target complex hasdissociated. The off-rate or dissociation rate of a slow off-rateaptamer, expressed as t_(1/2) values, can be about ≧30 min., ≧about 60min., ≧about 90 min., ≧about 120 min. ≧about 150 min. ≧about 180 min.≧about 210 min., and ≧about 240 min.

In one embodiment, a method for producing a synthetic library of nucleicacids comprises: 1) synthesizing the nucleic acids; 2) deprotecting thenucleic acids; 3) purifying the nucleic acids; and 4) analyzing thenucleic acids. In the synthesis step, a monomer mixture is preparedwhere the ratio of the various nucleotides in the mix is optimized toyield equal ratios of each nucleotide in the final product. One or moreof the monomers in the mixture may comprise a modified nucleotide.Amidite protection groups are used in this procedure and in oneembodiment, the monomer concentration is 0.1 M. During synthesis, thefive prime protecting group is retained in the product nucleic acid.Synthesis is conducted on a solid support (controlled pore glass, CPG)and at least about 80 cycles are completed to synthesize the finalproduct.

After the synthesis process, the nucleic acid product is deprotected. A1.0 M aqueous lysine buffer, pH 9.0 is employed to cleave apurinic siteswhile the product is retained on the support (controlled pore glass,CPG). These cleaved truncated sequences are washed away with deionized(dI) water two times. 500 μL of dI water are added after the two washesin preparation for the deprotection step. This step involves thetreatment with 1.0 mL of t-butylamine:methanol:water, 1:1:2, for 5 hoursat 70° C., followed by freezing, filtration, and evaporation to dryness.The nucleic acid product is purified based on the hydrophobicity of theprotecting group on a PRP-3 HPLC column (Hamilton). Appropriate columnfractions are collected and pooled, desalted, and evaporated to drynessto remove the volatile elution buffers. The final product is washed withwater by a centrifugation process and then re-suspended. Finally, theresuspended material is treated to deprotect the final product. Finalproduct is characterized by base composition, primer extension, andsequencing gel.

A candidate mixture of nucleic acids, or a library of nucleic acids, mayalso be produced by an enzymatic method using a solid phase. In oneembodiment, this method comprises the same basic steps described above.In this case the goal is the synthesis of an antisense library and theselibraries are produced with a 5′ biotin modification. All remainingsynthetic processes are as described above. Once the synthetic libraryis prepared, the nucleic acids may be used in a primer extension mixcontaining one or more modified nucleotides to produce the finalcandidate mixture in a classic primer extension method.

Aptamers may be synthesized by the same chemistry that is used for thesynthesis of a library. However, instead of a mixture of nucleotides,one nucleotide is introduced at each step in the synthesis to controlthe final sequence generated by routine methods. Modified nucleotidesmay be introduced into the synthesis process at the desired positions inthe sequence. Other functionalities may be introduced as desired usingknown chemical modifications of nucleotides.

As used herein, “candidate mixture” is a mixture of nucleic acids ofdiffering sequence from which to select a desired ligand. The source ofa candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. Modified nucleotides, such as nucleotides withphotoreactive groups or other modifications, can be incorporated intothe candidate mixture. In addition, a SELEX process can be used toproduce a candidate mixture, that is, a first SELEX process experimentcan be used to produce a ligand-enriched mixture of nucleic acids thatis used as the candidate mixture in a second SELEX process experiment. Acandidate mixture can also comprise nucleic acids with one or morecommon structural motifs. As used herein, a candidate mixture is alsosometimes referred to as a “pool” or a “library.” For example, an “RNApool” refers to a candidate mixture comprised of RNA.

In various embodiments, each nucleic acid in a candidate mixture mayhave fixed sequences on either side of a randomized region, tofacilitate the amplification process. The nucleic acids in the candidatemixture of nucleic acids can each further comprise fixed regions or“tail” sequences at their 5′ and 3′ termini to prevent the formation ofhigh molecular weight parasites during the amplification process.

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides of anylength, and such nucleotides may include deoxyribonucleotides,ribonucleotides, and/or analogs or chemically modifieddeoxyribonucleotides or ribonucleotides. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules.

If present, chemical modifications of a nucleotide can include, singlyor in any combination, 2′-position sugar modifications, 5-positionpyrimidine modifications (e.g.,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-[2-(1H-indole-3-yl)ethyl]carboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylammonium)propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-napthylcarboxyamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo- or 5-iodo-uracil, backbone modifications,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine, and the like. Modifications can alsoinclude 3′ and 5′ modifications, such as capping or pegylation. Othermodifications can include substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) andthose with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators, andthose with modified linkages (e.g., alpha anomeric nucleic acids, etc.).Further, any of the hydroxyl groups ordinarily present in a sugar may bereplaced by a phosphonate group or a phosphate group; protected bystandard protecting groups; or activated to prepare additional linkagesto additional nucleotides or to a solid support. The 5′ and 3′ terminalOH groups can be phosphorylated or substituted with amines, organiccapping group moieties of from about 1 to about 20 carbon atoms, ororganic capping group moieties of from about 1 to about 20 polyethyleneglycol (PEG) polymers or other hydrophilic or hydrophobic biological orsynthetic polymers. If present, a modification to the nucleotidestructure may be imparted before or after assembly of a polymer. Asequence of nucleotides may be interrupted by non-nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

In one embodiment, the variable region of the aptamer includesnucleotides that include modified bases. Certain modified aptamers maybe used in any of the described methods, devices, and kits. Thesemodified nucleotides have been shown to produce novel aptamers that havevery slow off-rates from their respective targets while maintaining highaffinity to the target. In one embodiment, the C-5 position of thepyrimidine bases may be modified. Aptamers containing nucleotides withmodified bases have a number of properties that are different than theproperties of standard aptamers that include only naturally occurringnucleotides (i.e., unmodified nucleotides). In one embodiment, themethod for modification of the nucleotides includes the use of an amidelinkage. However, other suitable methods for modification may be used.

Particular 5-position pyrimidine modifications include those describedin U.S. Pat. Nos. 5,719,273 and 5,945,527, as well as those illustratedin FIG. 14.

As used herein, “modified nucleic acid” refers to a nucleic acidsequence containing one or more modified nucleotides. In someembodiments it may be desirable that the modified nucleotides arecompatible with the SELEX process.

“Polypeptide,” “peptide,” and “protein” are used interchangeably hereinto refer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and/or it maybe interrupted by non-amino acids. The terms also encompass an aminoacid polymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Polypeptides can besingle chains or associated chains. “Marker” is used to describe atarget molecule, frequently a protein, that is a specific indicator orpredictor of a specific disease or condition for which a diagnosis isdesired.

As used herein, “photoreactive nucleotide” means any modified nucleotidethat is capable of photocrosslinking with a target, such as a protein,upon irradiation with certain wavelengths of light. For example,photoaptamers produced by the photoSELEX process can include aphotoreactive group selected from the following: 5-bromouracil (BrU),5-iodouracil (IU), 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil,4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine,8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine,8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine,8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuridine,8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine. A “photoreactive pyrimidine” means anymodified pyrimidine that is capable of photocrosslinking with a targetupon irradiation of certain wavelengths. Exemplary photoreactivepyrimidines include 5-bromo-uracil (BrdU), 5-bromo-cytosine (BrdC),5-iodo-uracil (IdU), and 5-iodo-cytosine (IdC). In various embodiments,the photoreactive functional group will absorb wavelengths of light thatare not absorbed by the target or the non-modified portions of theoligonucleotide.

“SELEX” refers to a process that combines the selection of nucleic acidsthat interact with a target in a desirable manner (e.g., binding to aprotein) with the amplification of those selected nucleic acids.Optional iterative cycling of the selection/amplification steps allowsselection of one or a small number of nucleic acids that interact moststrongly with the target from a pool that contains a very large numberof nucleic acids. Cycling of the selection/amplification procedure iscontinued until a selected goal is achieved. The SELEX methodology isdescribed in the SELEX patents. In some embodiments of the SELEXprocess, aptamers that bind non-covalently to their targets aregenerated. In other embodiments of the SELEX process, aptamers that bindcovalently to their targets are generated. In some embodiments thetargets used in the SELEX process are fixed in the same manner that ananalytical sample would be fixed during the use of the slow off-rateaptamer in the histological or cytological characterization of thatanalytical sample.

As used herein the term “amplification” or “amplifying” means anyprocess or combination of process steps that increases the amount ornumber of copies of a molecule or class of molecules.

“SELEX target” or “target molecule” or “target” refers herein to anycompound upon which a nucleic acid can act in a desirable manner. ASELEX target molecule can be a protein, peptide, nucleic acid,carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, pathogen, toxic substance, substrate,metabolite, transition state analog, cofactor, inhibitor, drug, dye,nutrient, growth factor, cell, tissue, any portion or fragment of any ofthe foregoing, etc., without limitation. Further the target may bemodified in one or more fashion. For example, proteins may be modifiedby glycosylation, phosphorylation, acetylation, phospholipids, and soforth. The target may be modified to different levels. Slow off-rateaptamers could be produced to differentiate the type or level ofmodification. In one embodiment, a SELEX target does not includemolecules that are known to bind nucleic acids, such as, for example,known nucleic acid binding proteins (e.g. transcription factors).Virtually any chemical or biological effector may be a suitable SELEXtarget. Molecules of any size can serve as SELEX targets. A target canalso be modified in certain ways to enhance the likelihood or strengthof an interaction between the target and the nucleic acid. A target canalso include any minor variation of a particular compound or molecule,such as, in the case of a protein, for example, minor variations inamino acid sequence, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component, which doesnot substantially alter the identity of the molecule. A “targetmolecule” or “target” is a set of copies of one type or species ofmolecule or multimolecular structure that is capable of binding to anaptamer. “Target molecules” or “targets” refer to more than one such setof molecules. Embodiments of the SELEX process in which the target is apeptide are described in U.S. Pat. No. 6,376,190, entitled “ModifiedSELEX Processes Without Purified Protein,” incorporated herein byreference in its entirety. FIG. 7 lists over 500 targets for whichaptamers have been produced including a variety of slow off-rateaptamers. The target may also be a “marker” or a molecule that isindicative of a specific disease state or condition and may be used inthe diagnosis of that specific disease state or for selection of anappropriate therapeutic regimen or as an indication of potentialtherapeutic efficacy. Examples of such markers include prostate specificantigen for prostate cancer, CMBK for heart disease, CEA, CA125 forcancer, HPV16 and HPV18 for cervical cancer, etc. An example of a markerthat is predictive of therapeutic efficacy would be HER2.

When a tissue SELEX process is used to identify target specific slowoff-rate aptamers from a tissue sample taken from a patient with a knowndisease, it may be of value to perform a secondary and optional counterselection. In this procedure the originally selected slow off-rateaptamers would be incubated with a tissue sample from a normal, ornon-diseased donor. Any slow off-rate aptamers in this pre-screenedcandidate mixture that reacted with the normal tissue would beeliminated from subsequent selection. This additional step would insurethat the final selected slow off-rate aptamer was highly specific. Thenormal tissue sample and the diseased tissue sample should be treated inthe same manner (i.e. fixation).

As used herein, “competitor molecule” and “competitor” are usedinterchangeably to refer to any molecule that can form a non-specificcomplex with a non-target molecule. In this context, non-targetmolecules include free aptamers, where, for example, a competitor can beused to inhibit the aptamer from binding (re-binding), non-specifically,to another non-target molecule. A “competitor molecule” or “competitor”is a set of copies of one type or species of molecule. “Competitormolecules” or “competitors” refer to more than one such set ofmolecules. Competitor molecules include, but are not limited tooligonucleotides, polyanions (e.g., heparin, herring sperm DNA, salmonsperm DNA, tRNA, dextran sulfate, polydextran, abasic phosphodiesterpolymers, dNTPs, and pyrophosphate). In various embodiments, acombination of one or more competitor can be used.

As used herein, “non-specific complex” refers to a non-covalentassociation between two or more molecules other than an aptamer and itstarget molecule. A non-specific complex represents an interactionbetween classes of molecules. Non-specific complexes include complexesformed between an aptamer and a non-target molecule, a competitor and anon-target molecule, a competitor and a target molecule, and a targetmolecule and a non-target molecule.

As used herein, the term “slow off-rate enrichment process” refers to aprocess of altering the relative concentrations of certain components ofa candidate mixture such that the relative concentration of aptameraffinity complexes having slow dissociation rates is increased relativeto the concentration of aptamer affinity complexes having faster, lessdesirable dissociation rates. In one embodiment, the slow off-rateenrichment process is a solution-based slow off-rate enrichment process.In this embodiment, a solution-based slow off-rate enrichment processtakes place in solution, such that neither the target nor the nucleicacids forming the aptamer affinity complexes in the mixture areimmobilized on a solid support during the slow off-rate enrichmentprocess. In various embodiments, the slow off-rate enrichment processcan include one or more steps, including the addition of an incubationwith a competitor molecule, dilution of the mixture, or a combination ofthese (e.g., dilution of the mixture in the presence of a competitormolecule). Because the effect of an slow off-rate enrichment processgenerally depends upon the differing dissociation rates of differentaptamer affinity complexes (i.e., aptamer affinity complexes formedbetween the target molecule and different nucleic acids in the candidatemixture), the duration of the slow off-rate enrichment process isselected so as to retain a high proportion of aptamer affinity complexeshaving slow dissociation rates while substantially reducing the numberof aptamer affinity complexes having fast dissociation rates. The slowoff-rate enrichment process may be used in one or more cycles during theSELEX process. When dilution and the addition of a competitor are usedin combination, they may be performed simultaneously or sequentially, inany order. The slow off-rate enrichment process can be used when thetotal target (protein) concentration in the mixture is low. In oneembodiment, when the slow off-rate enrichment process includes dilution,the mixture can be diluted as much as is practical, keeping in mind thatthe nucleic acids are recovered for subsequent rounds in the SELEXprocess. In one embodiment, the slow off-rate enrichment processincludes the use of a competitor as well as dilution, permitting themixture to be diluted less than might be necessary without the use of acompetitor.

In one embodiment, the slow off-rate enrichment process includes theaddition of a competitor, and the competitor is a polyanion (e.g.,heparin or dextran sulfate (dextran)). Heparin or dextran have been usedin the identification of specific aptamers in prior SELEX selections. Insuch methods, however, heparin or dextran is present during theequilibration step in which the target and aptamer bind to formcomplexes. In such methods, as the concentration of heparin or dextranincreases, the ratio of high affinity target/aptamer complexes to lowaffinity target/aptamer complexes increases. However, a highconcentration of heparin or dextran can reduce the number of highaffinity target/aptamer complexes at equilibrium due to competition fortarget binding between the nucleic acid and the competitor. By contrast,the presently described methods add the competitor after thetarget/aptamer complexes have been allowed to form and therefore doesnot affect the number of complexes formed. Addition of competitor afterequilibrium binding has occurred between target and aptamer creates anon-equilibrium state that evolves in time to a new equilibrium withfewer target/aptamer complexes. Trapping target/aptamer complexes beforethe new equilibrium has been reached enriches the sample for slowoff-rate aptamers since fast off-rate complexes will dissociate first.

In another embodiment, a polyanionic competitor (e.g., dextran sulfateor another polyanionic material) is used in the slow off-rate enrichmentprocess to facilitate the identification of an aptamer that isrefractory to the presence of the polyanion. In this context,“polyanionic refractory aptamer” is an aptamer that is capable offorming an aptamer/target complex that is less likely to dissociate inthe solution that also contains the polyanionic refractory material thanan aptamer/target complex that includes a non-polyanionic refractoryaptamer. In this manner, polyanionic refractory aptamers can be used inthe performance of analytical methods to detect the presence or amountor concentration of a target in a sample, where the detection methodincludes the use of the polyanionic material (e.g. dextran sulfate) towhich the aptamer is refractory.

Thus, in one embodiment, a method for producing a polyanionic refractoryaptamer is provided. In this embodiment, after contacting a candidatemixture of nucleic acids with the target the target and the nucleicacids in the candidate mixture are allowed to come to equilibrium. Apolyanionic competitor is introduced and allowed to incubate in thesolution for a period of time sufficient to insure that most of the fastoff-rate aptamers in the candidate mixture dissociate from the targetmolecule. Also, aptamers in the candidate mixture that may dissociate inthe presence of the polyanionic competitor will be released from thetarget molecule. The mixture is partitioned to isolate the highaffinity, slow off-rate aptamers that have remained in association withthe target molecule and to remove any uncomplexed materials from thesolution. The aptamer can then be released from the target molecule andisolated. The isolated aptamer can also be amplified and additionalrounds of selection applied to increase the overall performance of theselected aptamers. This process may also be used with a minimalincubation time if the selection of slow off-rate aptamers is not neededfor a specific application.

Thus, in one embodiment a modified SELEX process is provided for theidentification or production of aptamers having slow (long) off-rateswherein the target molecule and candidate mixture are contacted andincubated together for a period of time sufficient for equilibriumbinding between the target molecule and nucleic acids contained in thecandidate mixture to occur. Following equilibrium binding an excess ofcompetitor molecule, e.g., polyanion competitor, is added to the mixtureand the mixture is incubated together with the excess of competitormolecule for a predetermined period of time. A significant proportion ofaptamers having off-rates that are less than this predeterminedincubation period will dissociate from the target during thepredetermined incubation period. Re-association of these “fast” off-rateaptamers with the target is minimized because of the excess ofcompetitor molecule which can non-specifically bind to the target andoccupy aptamer binding sites on the target. A significant proportion ofaptamers having longer off-rates will remain complexed to the targetduring the predetermined incubation period. At the end of the incubationperiod, partitioning nucleic acid-target complexes from the remainder ofthe mixture allows for the separation of a population of slow off-rateaptamers from those having fast off-rates. A dissociation step can beused to dissociate the slow off-rate aptamers from their target andallows for isolation, identification, sequencing, synthesis andamplification of slow off-rate aptamers (either of individual aptamersor of a group of slow off-rate aptamers) that have high affinity andspecificity for the target molecule. As with conventional SELEX theaptamer sequences identified from one round of the modified SELEXprocess can be used in the synthesis of a new candidate mixture suchthat the steps of contacting, equilibrium binding, addition ofcompetitor molecule, incubation with competitor molecule andpartitioning of slow off-rate aptamers can be iterated/repeated as manytimes as desired.

The combination of allowing equilibrium binding of the candidate mixturewith the target prior to addition of competitor, followed by theaddition of an excess of competitor and incubation with the competitorfor a predetermined period of time allows for the selection of apopulation of aptamers having off-rates that are much greater than thosepreviously achieved.

In one embodiment a slow off-rate aptamer specific for a given target,is selected by the method comprising: (a) contacting the candidatemixture with a tissue or cell sample, wherein nucleic acids having anincreased affinity to the target relative to other nucleic acids in thecandidate mixture or other targets within the tissue or cell sample andmay be partitioned from the remainder of the candidate mixture and thetissue or cell sample; (b) partitioning the increased affinity and/orslow off-rate nucleic acids from the remainder of the candidate mixtureand the tissue or cell sample; and (c) amplifying the increasedaffinity, slow off-rate nucleic acids to yield a ligand-enriched mixtureof nucleic acids, whereby slow off-rate aptamers to the target moleculeare identified. Once a specific slow off-rate aptamer to the desiredtarget is selected it may be produced synthetically or through cloningor any other method for producing the specific nucleic acid sequence.Optionally a slow off-rate enrichment step may be used in the selectionprocess where the slow off-rate enrichment process may include dilutionof the candidate mixture in contact with the sample, introduction of acompetitor, or a combination of these methods. Optionally the tissue orcell sample is fixed prior to their use in the slow off-rate aptamerselection process. Optionally it may be desirable to confirm theidentity of the specific target selected by the tissue selection processby traditional biochemical isolation, purification, andcharacterization. These methods could include mass spectroscopy, 2-Delectrophoresis, etc. Further specificity could be introduced by theprocess called “counter-SELEX” that effectively discards ligands thathave ability to bind the target as well as closely related structuralanalogs of the target or targets within normal tissue or cell samples.During selection, the population of slow off-rate aptamers bound to thetarget is subjected to affinity elution with structural analogs ornormal tissue or cell samples and the sequences eluted are discarded.

To generate a slow off-rate aptamer to a cell or tissue target, the cellor tissue sample is first be mixed with a candidate mixture andequilibrium binding achieved. In order to achieve equilibrium binding,the candidate mixture is incubated with the target for at least about 5minutes, or at least about 15 minutes, about 30 minutes, about 45minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours,about 5 hours or about 6 hours. Once equilibrium binding is achieved theselection process may proceed.

The predetermined incubation period of competitor molecule with themixture of the candidate mixture and target molecule may be selected asdesired, taking account of factors such as the nature of the target andknown off-rates (if any) of known aptamers for the target. Predeterminedincubation periods may be chosen from: at least about 5 minutes, atleast about 10 minutes, at least about 20 minutes, at least about 30minutes, at least 45 about minutes, at least about 1 hour, at leastabout 2 hours, at least about 3 hours, at least about 4 hours, at leastabout 5 hours, at least about 6 hours.

In other embodiments a dilution is used as an off-rate enhancementprocess and incubation of the diluted candidate mixture, targetmolecule/aptamer complex may be undertaken for a predetermined period oftime, which may be chosen from: at least about 5 minutes, at least about10 minutes, at least about 20 minutes, at least about 30 minutes, atleast about 45 minutes, at least about 1 hour, at least about 2 hours,at least about 3 hours, at least about 4 hours, at least about 5 hours,at least about 6 hours.

Embodiments of the present disclosure are concerned with theidentification, production, synthesis and use of slow off-rate aptamersas well as uses of any specific aptamer. These are aptamers which have arate of dissociation (t_(1/2)) from a non-covalent aptamer-targetcomplex that is higher than that of aptamers normally obtained byconventional SELEX. For a mixture containing non-covalent complexes ofaptamer and target, the t_(1/2) represents the time taken for half ofthe aptamers to dissociate from the aptamer-target complexes. Thet_(1/2) of slow dissociation rate aptamers according to the presentdisclosure is chosen from one of: greater than or equal to about 30minutes; between about 30 minutes and about 240 minutes; between about30 minutes to about 60 minutes; between about 60 minutes to about 90minutes, between about 90 minutes to about 120 minutes; between about120 minutes to about 150 minutes; between about 150 minutes to about 180minutes; between about 180 minutes to about 210 minutes; between about210 minutes to about 240 minutes.

A characterizing feature of an aptamer identified by a SELEX procedureis its high affinity for its target. An aptamer will have a dissociationconstant (k_(d)) for its target that is chosen from one of: less thanabout 1 μM, less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 100 pM, less than about 10 pM, less thanabout 1 pM.

“Tissue target” or “tissue” refers herein to a certain subset of theSELEX targets described above. According to this definition, tissues aremacromolecules in a heterogeneous environment. As used herein, tissuerefers to a single cell type, a collection of cell types, an aggregateof cells, or an aggregate of macromolecules. This differs from simplerSELEX targets that are typically isolated soluble molecules, such asproteins. In some embodiments, tissues are insoluble macromolecules thatare orders of magnitude larger than simpler SELEX targets. Tissues arecomplex targets made up of numerous macromolecules, each macromoleculehaving numerous potential epitopes. The different macromolecules whichcomprise the numerous epitopes can be proteins, lipids, carbohydrates,etc., or combinations thereof. Tissues are generally a physical array ofmacromolecules that can be either fluid or rigid, both in terms ofstructure and composition. Extracellular matrix is an example of a morerigid tissue, both structurally and compositionally, while a membranebilayer is more fluid in structure and composition. Tissues aregenerally not soluble and remain in solid phase, and thus partitioningcan be accomplished relatively easily. Tissue includes, but is notlimited to, an aggregate of cells usually of a particular kind togetherwith their intercellular substance that form one of the structuralmaterials commonly used to denote the general cellular fabric of a givenorgan, e.g., kidney tissue, brain tissue. The four general classes oftissues are epithelial tissue, connective tissue, nerve tissue andmuscle tissue.

Examples of tissues which fall within this definition include, but arenot limited to, heterogeneous aggregates of macromolecules such asfibrin clots which are a cellular; homogeneous or heterogeneousaggregates of cells; higher ordered structures containing cells whichhave a specific function, such as organs, tumors, lymph nodes, arteries,etc.; and individual cells. Tissues or cells can be in their naturalenvironment, isolated, or in tissue culture. The tissue can be intact ormodified. The modification can include numerous changes such astransformation, transfection, activation, and substructure isolation,e.g., cell membranes, cell nuclei, cell organelles, etc.

Sources of the tissue, cell or subcellular structures can be obtainedfrom prokaryotes as well as eukaryotes. This includes human, animal,plant, bacterial, fungal, and viral structures.

As used herein, the term “labeling agent,” “label,” or “detectablemoiety,” or “detectable element” or “detectable component” refers to oneor more reagents that can be used to detect a target molecule/aptamercomplex. A detectable moiety or label is capable of being detecteddirectly or indirectly. In general, any reporter molecule that isdetectable can be a label. Labels include, for example, (i) reportermolecules that can be detected directly by virtue of generating asignal, (ii) specific binding pair members that may be detectedindirectly by subsequent binding to a cognate that contains a reportermolecule, (iii) mass tags detectable by mass spectrometry, (iv)oligonucleotide primers that can provide a template for amplification orligation, and (v) a specific polynucleotide sequence or recognitionsequence that can act as a ligand, such as, for example, a repressorprotein, wherein in the latter two instances the oligonucleotide primeror repressor protein will have, or be capable of having, a reportermolecule, and so forth. The reporter molecule can be a catalyst, such asan enzyme, a polynucleotide coding for a catalyst, promoter, dye,fluorescent molecule, quantum dot, chemiluminescent molecule, coenzyme,enzyme substrate, radioactive group, a small organic molecule,amplifiable polynucleotide sequence, a particle such as latex or carbonparticle, metal sol, crystallite, liposome, cell, etc., which may or maynot be further labeled with a dye, catalyst or other detectable group, amass tag that alters the weight of the molecule to which it isconjugated for mass spectrometry purposes, and the like. The label canbe selected from electromagnetic or electrochemical materials. In oneembodiment, the detectable label is a fluorescent dye. Other labels andlabeling schemes will be evident to one skilled in the art based on thedisclosure herein.

A detectable moiety (element or component) can include any of thereporter molecules listed above and any other chemical or component thatmay be used in any manner to generate a detectable signal. Thedetectable moiety, or signal generating label, may be detected via afluorescent signal, a chemiluminescent signal, or any other detectablesignal that is dependent upon the identity of the moiety. In the casewhere the detectable moiety is an enzyme (for example, alkalinephosphatase), the signal may be generated in the presence of the enzymesubstrate and any additional factors necessary for enzyme activity. Inthe case where the detectable moiety is an enzyme substrate, the signalmay be generated in the presence of the enzyme and any additionalfactors necessary for enzyme activity. Suitable reagent configurationsfor attaching the detectable moiety to a target molecule includecovalent attachment of the detectable moiety to the target molecule,non-covalent association of the detectable moiety with another labelingagent component that is covalently attached to the target molecule, andcovalent attachment of the detectable moiety to a labeling agentcomponent that is non-covalently associated with the target molecule.

Detectable moieties may be incorporated into an aptamer during synthesisby using labeled dNTPs, dyes that have been generated asphosphoramidites, or other chemistries that can be employed duringoligonucleotide synthesis, or may be incorporated by modification of thefinal aptamer product after synthesis. Each aptamer may include multipledetectable moieties to enhance signal generation. When multiple targetsfrom the same sample, for example a histological tissue section, are tobe detected then each target specific aptamer may be produced with anunique detectable moiety for simultaneous analysis of multiple targets.

As used herein, “partitioning” means any process whereby one or morecomponents of a mixture are separated from other components of themixture. For example, aptamers bound to target molecules can bepartitioned from other nucleic acids that are not bound to targetmolecules and from non-target molecules. More broadly stated,partitioning allows for the separation of all the nucleic acids in acandidate mixture into at least two pools based on their relativeaffinity and/or dissociation rate to the target molecule. Partitioningcan be accomplished by various methods known in the art, includingfiltration, affinity chromatography, liquid-liquid partitioning, HPLC,etc. For example, nucleic acid-protein pairs can be bound tonitrocellulose filters while unbound nucleic acids are not. Columns thatspecifically retain nucleic acid-target complexes can also be used forpartitioning. For example, oligonucleotides able to associate with atarget molecule bound on a column allow the use of column chromatographyfor separating and isolating the highest affinity aptamers. Beads uponwhich target molecules are conjugated can also be used to partitionaptamers in a mixture. If the beads are paramagnetic, the partitioningcan be achieved through application of a magnetic field. Surface plasmonresonance technology can be used to partition nucleic acids in a mixtureby immobilizing a target on a sensor chip and flowing the mixture overthe chip, wherein those nucleic acids having affinity for the target canbe bound to the target, and the remaining nucleic acids can be washedaway. Liquid-liquid partitioning can be used as well as filtration gelretardation and density gradient centrifugation. Affinity tags on thetarget molecules can also be used to separate nucleic acid moleculesbound to the tagged target from aptamers that are free in solution. Forexample, biotinylated target molecules, along with aptamers bound tothem, can be sequestered from the solution of unbound nucleic acidsequences using streptavidin paramagnetic beads. Affinity tags can alsobe incorporated into the aptamer during preparation. When Tissue SELEXis used to produce aptamers specific to one or more targets in abiological tissue (tissue section or cell preparation), the non-specificnucleic acids in a candidate mixture may be separated from the targetspecific aptamers by washing the tissue sample with one or more seriesof buffered reagents.

As used herein, “photoSELEX” is an acronym for Photochemical SystematicEvolution of Ligands by Exponential enrichment and refers to embodimentsof the SELEX process in which photocrosslinking aptamers are generated.In one embodiment of the photoSELEX process, a photoreactive nucleotideactivated by absorption of light is incorporated in place of a nativebase in either RNA- or in ssDNA-randomized oligonucleotide libraries,the nucleic acid target molecule mixture is irradiated causing somenucleic acids incorporated in nucleic acid-target molecule complexes tocrosslink to the target molecule via the photoreactive functionalgroups, and the selection step is a selection for photocrosslinkingactivity. The photoSELEX process is described in great detail in thePhotoSELEX patents.

As used herein, “photoaptamer” and “photoreactive aptamer” are usedinterchangeably to refer to an aptamer that contains one or morephotoreactive functional groups that can covalently bind to or“crosslink” with a target molecule. For example, a naturally occurringnucleic acid residue may be modified to include a chemical functionalgroup that confers photoreactivity upon the nucleic acid residue uponexposure to a radiation source of an appropriate wavelength. In someembodiments, a photoreactive aptamer is identified initially. In otherembodiments, an aptamer is first identified and is subsequently modifiedto incorporate one or more photoreactive functional groups, therebygenerating a photoaptamer. In these embodiments, one or morephotoreactive nucleic acid residues can be incorporated into an aptamereither by substituting a photoreactive nucleic acid residue in the placeof one or more other nucleotides, such as one or more of the thymidineand/or cytidine nucleotides in the aptamer, for example, or by modifyingone or more nucleic acid residues to include a photoreactive functionalgroup.

In yet other embodiments, certain nucleotides may be modified to produceslow off-rate aptamers that bind and form a covalent crosslink to theirtarget molecule in the affinity complex. This method encompasses slowoff-rate aptamers that bind and then may be linked to their targetmolecules. In various embodiments, the slow off-rate aptamers maycontain photoreactive groups that are capable of photocrosslinking tothe target molecule upon irradiation with light. In other embodiments,the slow off-rate aptamers are capable of bond formation with the targetin the absence of irradiation. A tight ionic interaction between theslow off-rate aptamer and target may also occur upon irradiation. Othermechanisms for chemical crosslinking may also be used. The crosslinkingof slow off-rate aptamer to its specific target maybe initiated by acrosslinking activator, such as irradiation, or a specific chemicalagent. In one embodiment, photocrosslinking occurs due to exposure toelectromagnetic radiation. Electromagnetic radiation includesultraviolet light, visible light, X-rays, and gamma rays. A crosslinkingstep may be added to the analysis of a tissue or cell sample at anypoint in the assay procedure.

A photoreactive group can be any chemical structure that contains aphotochromophore and that is capable of photocrosslinking with a targetmolecule. Although referred to herein as a photoreactive groups, in somecases, as described below, irradiation is not necessary for covalentbinding to occur between the slow off-rate aptamer and the target. Insome embodiments, the photoreactive group will absorb light of awavelength that is not absorbed by the target or the non-modifiedportions of the oligonucleotide. Photoreactive groups include5-halo-uridines, 5-halo-cytosines, 7-halo-adenosines,2-nitro-5-azidobenzoyls, diazirines, aryl azides, fluorinated arylazides, benzophenones, amino-benzophenones, psoralens, anthraquinones,etc.

Exemplary photoreactive functional groups that may be incorporated by aphotoaptamer include 5-bromouracil, 5-iodouracil, 5-bromovinyluracil,5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-thiouracil,4-thiocytosine, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine,5-iodovinylcyto sine, 5-azidocyto sine, 8-azidoadenine, 8-bromoadenine,8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine,8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine,8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine,5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil,7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine,and 7-deaza-7-bromoguanine.

In addition to these exemplary nucleoside-based photoreactive functionalgroups, other photoreactive functional groups that can be added to aterminal end of an aptamer using an appropriate linker molecule can alsobe used. Such photoreactive functional groups include benzophenone,anthraquinone, 4-azido-2-nitro-aniline, psoralen, derivatives of any ofthese, and the like.

A photoreactive functional group incorporated by a photoaptamer may beactivated by any suitable method. In one embodiment, a photoaptamercontaining a photoreactive functional group can be crosslinked to itstarget by exposing the photoaptamer and its bound target molecule to asource of electromagnetic radiation. Suitable types of electromagneticradiation include ultraviolet light, visible light, X-rays, and gammarays. Suitable radiation sources include sources that utilize eithermonochromatic light or filtered polychromatic light.

As used herein, the term “the affinity SELEX process” refers toembodiments of the SELEX process in which non-photocrosslinking aptamersto targets are generated. In some embodiments of the affinity SELEXprocess, the target is immobilized on a solid support either before orafter the target is contacted with the candidate mixture of nucleicacids. The association of the target with the solid support allowsnucleic acids in the candidate mixture that have bound and in the casewhere a slow off-rate enrichment process is used, stay bound to thetarget to be partitioned from the remainder of the candidate mixture.The term “bead affinity SELEX process” refers to particular embodimentsof the affinity SELEX process where the target is immobilized on a bead,for example, before contact with the candidate mixture of nucleic acids.In some embodiments, the beads are paramagnetic beads. The term “filteraffinity SELEX process” refers to embodiments where nucleic acid targetcomplexes are partitioned from candidate mixture by virtue of theirassociation with a filter, such as a nitrocellulose filter. Thisincludes embodiments where the target and nucleic acids are initiallycontacted in solution, and contacted with the filter, and also includesembodiments where nucleic acids are contacted with target that ispre-immobilized on the filter. The term “plate affinity SELEX process”refers to embodiments where the target is immobilized on the surface ofa plate, such as, for example, a multi-well microtiter plate. In someembodiments, the plate is comprised of polystyrene. In some embodiments,the target is attached to the plate in the plate affinity SELEX processthrough hydrophobic interactions.

The present disclosure describes improved SELEX methods for generatingand using aptamers that are capable of binding to target molecules. Morespecifically, the present disclosure describes methods for identifyingaptamers and/or photoaptamers having slower rates of dissociation fromtheir respective targeted molecules than aptamers obtained with previousSELEX methods. The disclosure further describes aptamers and/orphotoaptamers obtained using the methods described herein and methods ofusing the same.

In one embodiment, a method is provided for identifying an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising (a) preparing a candidate mixture of nucleic acid sequences;(b) contacting the candidate mixture with a target molecule whereinnucleic acids with the highest relative affinities to the targetmolecule preferentially bind the target molecule, forming nucleicacid-target molecule complexes; (c) applying a slow off-rate enrichmentprocess to allow the dissociation of nucleic acid-target moleculecomplexes with relatively fast dissociation rates; (d) partitioning theremaining nucleic acid-target molecule complexes from both free nucleicacids and non-target molecules in the candidate mixture; and (e)identifying an aptamer to the target molecule. The process may furtherinclude the iterative step of amplifying the nucleic acids that bind tothe target molecule to yield a mixture of nucleic acids enriched insequences that are able to bind to the target molecule yet producenucleic acid-target molecule complexes having slow dissociation rates.As defined above, the slow off-rate enrichment process can be selectedfrom (a) diluting the candidate mixture containing the nucleicacid-target molecule complexes; (b) adding at least one competitor tothe candidate mixture containing the nucleic acid-target moleculecomplexes, and diluting the candidate mixture containing the nucleicacid-target molecule complexes; (c) and adding at least one competitorto the candidate mixture containing the nucleic acid-target moleculecomplexes.

In one embodiment, a method is provided for producing an aptamer havinga slow rate of dissociation from its target molecule, the methodcomprising (a) preparing a candidate mixture of nucleic acid sequences;(b) contacting the candidate mixture with a target molecule whereinnucleic acids with the highest relative affinities to the targetmolecule preferentially bind the target molecule, forming nucleicacid-target molecule complexes; (c) applying a slow off-rate enrichmentprocess to allow the dissociation of nucleic acid-target moleculecomplexes with relatively fast dissociation rates; (d) partitioning theremaining nucleic acid-target molecule complexes from both free nucleicacids and non-target molecules in the candidate mixture; and (e)producing an aptamer to the target molecule. The process may furtherinclude the iterative step of amplifying the nucleic acids that bind tothe target molecule to yield a mixture of nucleic acids enriched insequences that are able to bind to the target molecule yet producenucleic acid-target molecule complexes having slow dissociation rates.As defined above, the slow off-rate enrichment process can be selectedfrom (a) diluting the candidate mixture containing the nucleicacid-target molecule complexes; (b) adding at least one competitor tothe candidate mixture containing the nucleic acid-target moleculecomplexes, and diluting the candidate mixture containing the nucleicacid-target molecule complexes; (c) and adding at least one competitorto the candidate mixture containing the nucleic acid-target moleculecomplexes.

In one embodiment, a method is provided for identifying an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising: (a) preparing a candidate mixture of nucleic acids; (b)contacting the candidate mixture with a target molecule, wherein nucleicacids having an increased affinity to the target molecule relative toother nucleic acids in the candidate mixture bind the target molecule,forming nucleic acid-target molecule complexes; (c) incubating thecandidate mixture and target molecule together for a period of timesufficient to achieve equilibrium binding; (d) applying a slow off-rateenrichment process to allow the dissociation of nucleic acid-targetmolecule complexes with relatively fast dissociation rates to themixture of (c); (e) incubating the mixture of the candidate mixture, thenucleic acid-target molecule complexes and the competitor molecule from(d) for a predetermined period of time; (f) partitioning the nucleicacid-target molecule complexes from the candidate mixture; (g)dissociating the nucleic acid-target molecule complexes to generate freenucleic acids; (h) amplifying the free nucleic acids to yield a mixtureof nucleic acids enriched in nucleic acid sequences that are capable ofbinding to the target molecule with increased affinity, whereby anaptamer to the target molecule may be identified. As defined above, theslow off-rate enrichment process can be selected from (a) diluting thecandidate mixture containing the nucleic acid-target molecule complexes;(b) adding at least one competitor to the candidate mixture containingthe nucleic acid-target molecule complexes, and diluting the candidatemixture containing the nucleic acid-target molecule complexes; (c) andadding at least one competitor to the candidate mixture containing thenucleic acid-target molecule complexes.

In another embodiment, a method is provided for producing an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising: (a) preparing a candidate mixture of nucleic acids; (b)contacting the candidate mixture with a target molecule, wherein nucleicacids having an increased affinity to the target molecule relative toother nucleic acids in the candidate mixture bind the target molecule,forming nucleic acid-target molecule complexes; (c) incubating thecandidate mixture and target molecule together for a period of timesufficient to achieve equilibrium binding; (d) applying a slow off-rateenrichment process to allow the dissociation of nucleic acid-targetmolecule complexes with relatively fast dissociation rates to themixture of (c); (e) incubating the mixture of the candidate mixture, thenucleic acid-target molecule complexes and the competitor molecule from(d) for a predetermined period of time; (f) partitioning the nucleicacid-target molecule complexes from the candidate mixture; (g)dissociating the nucleic acid-target molecule complexes to generate freenucleic acids; (h) amplifying the free nucleic acids to yield a mixtureof nucleic acids enriched in nucleic acid sequences that are capable ofbinding to the target molecule with increased affinity, whereby anaptamer to the target molecule may be produced. As defined above, theslow off-rate enrichment process can be selected from (a) diluting thecandidate mixture containing the nucleic acid-target molecule complexes;(b) adding at least one competitor to the candidate mixture containingthe nucleic acid-target molecule complexes, and diluting the candidatemixture containing the nucleic acid-target molecule complexes; (c) andadding at least one competitor to the candidate mixture containing thenucleic acid-target molecule complexes.

In another embodiment, a method is provided of identifying an aptamerhaving a slow rate of dissociation from its target molecule, the methodcomprising: (a) preparing a candidate mixture of nucleic acids, whereinthe candidate mixture comprises modified nucleic acids in which one,several or all pyrimidines in at least one, or each, nucleic acid of thecandidate mixture is chemically modified at the 5-position; (b)contacting the candidate mixture with a target molecule, wherein nucleicacids having an increased affinity to the target molecule relative toother nucleic acids in the candidate mixture bind the target molecule,forming nucleic acid-target molecule complexes; (c) partitioning theincreased affinity nucleic acids from the remainder of the candidatemixture; and (d) amplifying the increased affinity nucleic acids toyield a mixture of nucleic acids enriched in nucleic acid sequences thatare capable of binding to the target molecule with increased affinity,whereby an aptamer to the target molecule may be identified.

In another embodiment, a method is provided for producing an aptamerhaving a slow rate of dissociation from its target molecule, said methodcomprising preparing or synthesizing an aptamer that includes a nucleicacid sequence identified by the following process: (a) preparing acandidate mixture of nucleic acids, wherein the candidate mixturecomprises modified nucleic acids in which one, several or allpyrimidines in at least one, or each, nucleic acid of the candidatemixture is chemically modified at the 5-position; (b) contacting thecandidate mixture with a target molecule, wherein nucleic acids havingan increased affinity to the target molecule relative to other nucleicacids in the candidate mixture bind the target molecule, forming nucleicacid-target molecule complexes; (c) partitioning the increased affinitynucleic acids from the remainder of the candidate mixture; and (d)amplifying the increased affinity nucleic acids to yield a mixture ofnucleic acids enriched in nucleic acid sequences that are capable ofbinding to the target molecule with increased affinity, whereby anaptamer to the target molecule is identified.

In another embodiment, a non-covalent complex of an aptamer and itstarget is provided, wherein the rate of dissociation (t_(1/2)) of theaptamer from the target is chosen from one of: greater than or equal toabout 30 minutes; between about 30 minutes and about 240 minutes; about30 minutes to about 60 minutes; about 60 minutes to about 90 minutes;about 90 minutes to about 120 minutes; about 120 minutes to about 150minutes; about 150 minutes to about 180 minutes; about 180 minutes toabout 210 minutes; about 210 minutes to about 240 minutes.

In another embodiment, a non-covalent complex of an aptamer and a targetis provided, wherein the aptamer has a K_(d) for the target of about 100nM or less, wherein the rate of dissociation (t_(1/2)) of the aptamerfrom the target is greater than or equal to about 30 minutes, andwherein one, several or all pyrimidines in the nucleic acid sequence ofthe aptamer are modified at the 5-position of the base. Themodifications may be selected from the group of compounds shown in FIG.14, these modifications are referred to as “base modified nucleotides”.Aptamers may be designed with any combination of the base modifiedpyrimidines desired. Improved methods for performing SELEX with modifiednucleotides, including nucleotides which contain photoactive groups ornucleotides which contain placeholders for photoactive groups aredisclosed in U.S. application Ser. No. 12/175,388, entitled “ImprovedSELEX and PHOTOSELEX” which is being filed concurrently with the instantapplication and which is incorporated herein by reference in itsentirety. In another embodiment, the candidate mixture of nucleic acidmolecules includes nucleic acids containing modified nucleotide basesthat may aid in the formation of modified nucleic acid-target complexeswith relatively slow dissociation rates.

The various methods and steps described herein can be used to generatean aptamer capable of either (1) binding to a target molecule or (2)binding to a target molecule and subsequently forming a covalent linkagewith the target molecule upon irradiation.

Aptamers identified according to the methods described herein are usefulin a range of diagnostic and therapeutic methods. Slow off-rate aptamerswill bind to the target for a longer duration. This is useful indiagnostic methods where the binding of an aptamer to the target may beused to detect the presence, absence, amount or quantity of the targetmolecule and a prolonged interaction of the aptamer and targetfacilitates such detection. A similar advantage may be afforded whereslow off-rate aptamers are used in imaging methods, in vitro or in vivo.A prolonged interaction of aptamer and target also provides for improvedtherapeutic methods of treatment where the prolonged interaction mayallow for an improved therapeutic effect, e.g. owing to the longeractivation or inhibition of the target molecule or downstream signalingcascade. These slow off-rate aptamers and aptamers with high affinitymay be used in cytological and histological molecular detection andidentification methods.

Accordingly, in various embodiments, slow off-rate aptamers obtained,identified or produced by the described methods can be used in a varietyof methods of medical treatment or methods of diagnosis (in vitro or invivo). In one embodiment, slow off-rate aptamers can be used in a methodof treatment of disease. In one embodiment, slow off-rate aptamers canbe used in a method for diagnosis of disease in vivo. In anotherembodiment, slow off-rate aptamers can be used in vitro for thediagnosis of disease. In another embodiment, a slow off-rate aptamer canbe used in the manufacture of a therapeutic (e.g. pharmaceuticalcomposition) or the manufacture of a diagnostic agent for use in amethod of treatment or diagnosis of disease. Diagnostic or therapeuticapplications of slow off-rate aptamers may involve a diagnostic ortherapeutic outcome that depends on the specific and/or high affinitybinding of the slow off-rate aptamer to its target. Slow off-rateaptamers may also be used in target validation and high throughputscreening assays in the drug development process.

In one embodiment, slow off-rate aptamers are suitable reagents formolecular imaging in vivo. In this embodiment, a slow off-rate aptamermay be used in vivo to detect the presence of a pathology, diseaseprocess, or other condition in the body of an individual (e.g., a humanor an animal), where the binding of the aptamer to its target indicatesthe presence of the disease process or other condition. For example, anaptamer to the VEGF receptor may be used in vivo to detect the presenceof cancer in a particular area (e.g., a tissue, an organ, etc.) of thebody of an individual, as the VEGF receptor is abundantly expressedwithin tumors and their neovasculature, or an aptamer to the EGFreceptor may be used in vivo to detect the presence of cancer in aparticular area (e.g., a tissue, an organ, etc.) of the body of anindividual, as the EGF receptor is often expressed at high levels ontumor cells. That is, the molecular target will be the extracellulardomain (ECD) of an induced receptor, as such targets are located outsideof the cells and are accessible through the vasculature. Additionally,the ECDs tend to be localized at the site of pathology, even though somesmall fraction of the specific ECD may be shed through biologicalprocesses, including cell death.

The obvious candidates for molecular imaging, high affinity monoclonalantibodies, have not become the reagent of choice for this application.Molecular imaging reagents have precise requirements. They must havehigh binding activity for their intended target, and low bindingactivity for other targets in a human or animal. Slow off-rate aptamershave unique advantages that render them desirable for use in molecularimaging in vivo. On the one hand, they are selected to have slowdissociation rate constants, thus allowing residence in vivo on theintended target for a substantial length of time (at least about 30minutes). On the other hand, slow off-rate aptamers are expected to havevery fast clearance from the vasculature. Slow dissociation rateconstants and fast clearance from the vasculature are two desiredproperties for molecular imaging in vivo. From a kinetic prospective,good in vivo molecular imaging reagents must stay localized at the siteof the pathology while the free reagent concentration in the surroundingvasculature becomes low. This is a signal-to-noise constraint. Suitablesignal-to-noise ratios may be obtained by accumulation of signal at thesite of pathology in excess of the signal in the vasculature, or may beobtained by retention of a signal at the site of the pathology while thevasculature concentration is diminished.

Aptamers that do not have slow off-rate properties, of about the samemolecular weight and net charge as slow off-rate aptamers, have beenstudied in animals and humans for more than a decade. Generally, it hasbeen found that these aptamers clear from the vasculature quickly,usually by entering the kidney and/or the liver and then being furthermetabolized for excretion. Such aptamers show so-called “first pass”clearance unless high molecular weight adducts (such as, for example,PEG) are linked to the aptamers. Experiments have been done with anaptamer whose target is tenascin C, an extracellular protein (not anECD) found at high concentrations in some tumors. In those experiments,the tenascin C-specific aptamer cleared quickly and was able to beretained at the site of the tumor because the extracellular localconcentration of tenascin C is very high. Slow off-rate aptamers, bycontrast, will maintain the fast clearance rate of aptamers but offer akinetic advantage due to their slow dissociation rates, rendering themsuitable for use with targets whose presence at the site of interest(e.g., the site of pathology) may be somewhat sparse (ECDs on tumors,for example).

Alternative reagents for molecular imaging do not share the two slowoff-rate aptamer properties (i.e., slow dissociation rate and fastclearance from the body). Monoclonal antibodies often have high affinityand specificity, and may have slow dissociation rate constants; however,monoclonal antibodies have very slow clearance rates from thevasculature. Short peptides, identified through, for example, phagedisplay, may have fast clearance but poor affinity and specificity andfast dissociation rates from their intended targets. Affibodies, aparticular peptide version of an antibody mimetic, may have reasonableaffinity and specificity and may have faster clearance than monoclonalantibodies, yet in order to achieve slow dissociation rates from theirtargets, affibodies are often made into dimers and higher ordermultimers, slowing their clearance at the same time that theirdissociation rates are enhanced.

Slow off-rate aptamers may be used for molecular imaging in vivo withone or more low molecular weight adducts to both protect the slowoff-rate aptamer from nucleases in the body and detect the intendedtarget once bound by the slow off-rate aptamer. For example, slowoff-rate aptamers may be attacked by nucleases in the blood, typicallyexonucleases (for DNA) that are easily blocked by using exonucleaserefractive adducts at the 5′ and 3′ terminal positions of the slowoff-rate aptamer, or endonucleases (for RNA) that are easily blocked byincorporating endonuclease refractive pyrimidines (such as, for example,2′ fluoro nucleotides) in the slow off-rate aptamer. Detection of theslow off-rate aptamer-target complex may be achieved by attaching adetection moiety to the slow off-rate aptamer. In some embodiments, thedetection moiety for these purposes may include cages for radioactivemolecules (e.g., technetium 99), clusters of iron for magnetic resonancedetection, isotopes of fluorine for PET imaging, and the like. Themodifications made to the slow off-rate aptamer to protect the integrityof the slow off-rate aptamer in the body and enable detection of theintended target should be designed such that they do not interfere withthe slow off-rate aptamer's interaction with its target and do not causethe slow off-rate aptamer to clear too slowly from the vasculature.

Diagnostic or assay devices, e.g. columns, test strips or biochips,having one or more slow off-rate aptamers adhered to a solid surface ofthe device are also provided. The aptamer(s) may be positioned so as tobe capable of binding target molecules that are contacted with the solidsurface to form aptamer-target complexes that remain adhered to thesurface of the device, thereby capturing the target and enablingdetection and optionally quantitation of the target. An array of slowoff-rate aptamers (which may be the same or different) may be providedon such a device.

In another embodiment, complexes including a slow off-rate aptamer and atarget molecule are provided. In other embodiments, a class of aptamerscharacterized by having high affinity for their corresponding targetmolecules and slow dissociation rates (t_(1/2)) from a non-covalentcomplex of the aptamer and target is provided.

The basic SELEX process generally begins with the preparation of acandidate mixture of nucleic acids of differing sequence. The candidatemixture generally includes nucleic acid sequences that include two fixedregions (i.e., each of the members of the candidate mixture contains thesame sequences in the same location) and a variable region. Typically,the fixed sequence regions are selected such that they assist in theamplification steps described below, or enhance the potential of a givenstructural arrangement of the nucleic acids in the candidate mixture.The variable region typically provides the target binding region of eachnucleic acid in the candidate mixture, and this variable region can becompletely randomized (i.e., the probability of finding a base at anyposition being one in four) or only partially randomized (e.g., theprobability of finding a base at any location can be selected at anylevel between 0 and 100 percent). The prepared candidate mixture iscontacted with the selected target under conditions that are favorablefor binding to occur between the target and members of the candidatemixture. Under these conditions, the interaction between the target andthe nucleic acids of the candidate mixture generally forms nucleicacid-target pairs that have the strongest relative affinity betweenmembers of the pair. The nucleic acids with the highest affinity for thetarget are partitioned from those nucleic acids with lesser affinity tothe target. The partitioning process is conducted in a manner thatretains the maximum number of high affinity candidates. Those nucleicacids selected during partitioning as having a relatively high affinityto the target are amplified to create a new candidate mixture that isenriched in nucleic acids having a relatively high affinity for thetarget. By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acidmixture to the target will generally increase. Taken to its extreme, theSELEX process will yield a candidate mixture containing one or a verysmall number of unique nucleic acids representing those nucleic acidsfrom the original candidate mixture that have the highest affinity tothe target molecule. However, this basic SELEX process does not selectfor aptamers that have slow off-rates from their targets.

The SELEX patents and the PhotoSELEX patents describe and elaborate onthis process in great detail. These patents include descriptions of thevarious targets that can be used in the process; methods for thepreparation of the initial candidate mixture; methods for partitioningnucleic acids within a candidate mixture; and methods for amplifyingpartitioned nucleic acids to generate enriched candidate mixtures. TheSELEX patents also describe aptamer solutions obtained to a number ofdifferent types of target molecules, including protein targets whereinthe protein is and is not a nucleic acid binding protein. With referenceto FIG. 2 the modified SELEX process disclosed herein includes theintroduction of a slow off-rate enrichment process followingequilibration of the candidate mixture of nucleic acids with the targetor targets and a partitioning step prior to subsequent steps in theSELEX process. Introduction of a slow off-rate enrichment process to thebasic SELEX process provides a means for enrichment of aptamer affinitycomplexes with slow dissociation rates from a set of nucleic acid-targetcomplexes that includes a variety of dissociation rates. Thus, themodified SELEX process provides a method for identifying aptamers thatbind target molecules and, once bound, have relatively slow rates ofdissociation (also referred to herein as “off-rates”) from the targetmolecule.

As used herein “binding” generally refers to the formation of anon-covalent association between the ligand and the target, althoughsuch binding is not necessarily reversible. The terms “nucleicacid-target complex” or “complex” or “affinity complex” are used torefer to the product of such non-covalent binding association.

In various embodiments, the slow off-rate aptamers can be single- ordouble-stranded RNA or DNA oligonucleotides. The aptamers can containnon-standard or modified bases. Further, the aptamers can contain anytype of modification. As used herein, a “modified base” may include arelatively simple modification to a natural nucleic acid residue, whichmodification confers a change in the physical properties of the nucleicacid residue. Such modifications include, but are not limited to,modifications at the 5-position of pyrimidines, substitution withhydrophobic groups, e.g., benzyl, iso-butyl, indole, or napthyl, orsubstitution with hydrophilic groups, e.g., quaternary amine orguanidinium, or more “neutral” groups, e.g., imidazole and the like.Additional modifications may be present in the ribose ring, e.g.,2′-position, such as 2′-amino (2′-NH₂) and 2′-fluoro (2′-F), or thephosphodiester backbone, e.g., phosphorothioates or methyl phosphonates.

In various embodiments, a candidate mixture containing a randomized setof nucleic acid sequences containing modified nucleotide bases is mixedwith a quantity of the target molecule and allowed to establish bindingequilibrium with the target molecule. Generally, only some of thosenucleic acids that bind with high affinity to the target molecule willefficiently partition with the target.

In various embodiments, the candidate mixture includes nucleic acidsequences having variable regions that include modified groups. Themodified groups can be modified nucleotide bases. The variable regioncan contain fully or partially random sequences; it can also containsub-portions of a fixed sequence that is incorporated within thevariable region. The nucleotides within the fixed regions can alsocontain modified nucleotide bases, or they can contain the standard setof naturally occurring bases.

In some embodiments, amplification occurs after members of the testmixture have been partitioned, and it is the nucleic acid that isamplified. For example, amplifying RNA molecules can be carried out by asequence of three reactions: making cDNA copies of selected RNAs, usingthe polymerase chain reaction to increase the copy number of each cDNA,and transcribing the cDNA copies to obtain RNA molecules having the samesequences as the selected RNAs. Any reaction or combination of reactionsknown in the art can be used as appropriate, including direct DNAreplication, direct RNA amplification and the like, as will berecognized by those skilled in the art. The amplification method mayresult in the proportions of the amplified mixture being representativeof the proportions of different sequences in the mixture prior toamplification. It is known that many modifications to nucleic acids arecompatible with enzymatic amplification. Modifications that are notcompatible with amplification can be made after each round ofamplification, if necessary.

The nucleic acid candidate mixture can be modified in various ways toenhance the probability of the nucleic acids having facilitatingproperties or other desirable properties, particularly those thatenhance the interaction between the nucleic acid and the target.Contemplated modifications include modifications that introduce otherchemical groups that have the correct charge, polarizability, hydrogenbonding, or electrostatic interaction to enhance the desiredligand-target interactions. The modifications that may enhance thebinding properties, including the affinity and/or dissociation rates, ofthe nucleic acid, for example, include hydrophilic moieties, hydrophobicmoieties, rigid structures, functional groups found in proteins such asimidazoles, primary alcohols, carboxylates, guanidinium groups, aminogroups, thiols and the like. Modifications can also be used to increasethe survival of aptamer-target complexes under stringent selectionpressures that can be applied to produce slow off-rate aptamers to awide range of targets. In one embodiment, Bz-dU(benzyl-dU) is used inthe generation of the candidate mixtures used to produce slow off-rateaptamers, although other modified nucleotides are well suited to theproduction of such aptamers. Other modified nucleotides are shown inFIG. 14. A modified nucleotide candidate mixture for the purpose of thisapplication is any RNA or DNA candidate mixture that includes bothnaturally occurring and other than the naturally occurring nucleotides.Suitable modifications include modifications on every residue of thenucleic acid, on a single residue of the nucleic acid, on randomresidues, on all pyrimidines or all purines, on all occurrences of aspecific base (i.e., G, C, A, T or U) in the nucleic acid, or any othermodification scheme that may be suitable for a particular application.It is recognized that modification is not a prerequisite forfacilitating activity or binding ability of the aptamers. Aptamers mayinclude modified dUTP and dCTP residues.

Candidate mixtures for slow off-rate aptamers may comprise a set ofpyrimidines having a different modification at the C-5 base position.The C-5 modification may be introduced through an amide linkage,directly, or indirectly, or through another type of linkage. Thesecandidate mixtures are used in a SELEX process to identify slow off-rateaptamers. This process may be also include the use of the slow off-rateenrichment process. Candidate mixtures may be produced enzymatically orsynthetically.

As described above, the nucleotides can be modified in any number ofways, including modifications of the ribose and/or phosphate and/or basepositions. Certain modifications are described in U.S. Pat. No.5,660,985 entitled “High Affinity Nucleic Acid Ligands ContainingModified Nucleotides,” U.S. Pat. No. 5,428,149 entitled “Method forPalladium Catalyzed Carbon-Carbon Coupling and Products,” U.S. Pat. No.5,580,972 entitled “Purine Nucleoside Modifications by PalladiumCatalyzed Methods,” all of which are incorporated by reference herein.In one embodiment, modifications are those wherein another chemicalgroup is attached to the 5-position of a pyrimidine or the 2′ positionof a sugar. There is no limitation on the type of other chemical groupthat can be incorporated on the individual nucleotides. In someembodiments, the resulting modified nucleotide is amplifiable or can bemodified subsequent to the amplification steps (see, e.g., U.S. Pat. No.6,300,074 entitled “Systematic evolution of ligands by exponentialenrichment: Chemi-SELEX”.

In yet other embodiments, certain nucleotides are modified to produceaptamers that bind and form a covalent crosslink to their targetmolecule upon photo-activation of the affinity complex. This methodencompasses aptamers that bind, photocrosslink, and/or photoinactivatetarget molecules. In various embodiments, the aptamers containphotoreactive groups that are capable of photocrosslinking to the targetmolecule upon irradiation with light. In other embodiments, the aptamersare capable of bond formation with the target in the absence ofirradiation.

A photoreactive group can be any chemical structure that contains aphotochromophore and that is capable of photocrosslinking with a targetmolecule. Although referred to herein as a photoreactive group, in somecases, as described below, irradiation is not necessary for covalentbinding to occur between the aptamer and the target. In someembodiments, the photoreactive group will absorb light of a wavelengththat is not absorbed by the target or the non-modified portions of theoligonucleotide. Photoreactive groups include 5-halo-uridines,5-halo-cytosines, 7-halo-adenosines, 2-nitro-5-azidobenzoyls,diazirines, aryl azides, fluorinated aryl azides, benzophenones,amino-benzophenones, psoralens, anthraquinones, etc.

The photoreactive groups generally form bonds with the target uponirradiation of the associated nucleic acid-target pair. In some cases,irradiation is not required for bond formation to occur. Thephotocrosslink that typically occurs will be the formation of a covalentbond between the associated aptamer and the target. However, a tightionic interaction between the aptamer and target may also occur uponirradiation.

In one embodiment, photocrosslinking occurs due to exposure toelectromagnetic radiation. Electromagnetic radiation includesultraviolet light, visible light, X-ray, and gamma ray.

In various other embodiments, a limited selection of oligonucleotidesusing a SELEX method is followed by selection using a photoSELEX method.The initial SELEX selection rounds are conducted with oligonucleotidescontaining photoreactive groups. After a number of SELEX rounds,photoSELEX is conducted to select oligonucleotides capable of bindingthe target molecule. In another embodiment, the production of an aptamerthat includes a cleavable or releasable section (also described as anelement or component) in the aptamer sequence is described. Theseadditional components or elements are structural elements or componentsthat introduce additional functionality into the aptamer and are thusfunctional elements or components. The aptamer is further produced withone or more of the following additional components (also described as afunctional or structural element or component or moiety in anycombination of these terms): a labeled or detectable component, a spacercomponent, and a specific binding tag or immobilization element orcomponent.

As noted above, the present disclosure provides methods for identifyingaptamers that bind target molecules and once bound have slow rates ofdissociation or off-rates. The slow off-rates obtained with this methodcan exceed a half-life of about one hour and as much as about 240minutes, that is, once a set of nucleic acid-target complexes isgenerated, half of the complexes in the set remain bound after one hour.Because the effect of a slow off-rate enrichment process depends uponthe differing dissociation rates of aptamer affinity complexes, theduration of the slow off-rate enrichment process is chosen so as toretain a high proportion of aptamer affinity complexes with slowdissociation rates while substantially reducing the number of aptameraffinity complexes with fast dissociation rates. For example, incubatingthe mixture for relatively longer periods of time after imposing theslow off-rate enrichment process will select for aptamers with longerdissociation rates than aptamers selected using slow off-rate enrichmentprocess having shorter incubation periods.

In various embodiments, the candidate mixture is mixed with a quantityof the target molecule and allowed to establish binding equilibrium withthe target molecule. Prior to partitioning the target bound nucleicacids from those free in solution, a slow off-rate enrichment process isimposed to enrich the bound population for slow dissociation rates. Asnoted above, the slow off-rate enrichment process can be applied by theaddition of a competitor molecule, by sample dilution, by a combinationof sample dilution in the presence of a competitor molecule. Thus, inone embodiment, the slow off-rate enrichment process is applied byintroducing competitor molecules into the mixture containing the nucleicacid-target complexes and incubating the mixture for some period of timebefore partitioning free from bound nucleic acids. The amount ofcompetitor molecules is generally at least one order of magnitude higherthan that of the nucleic acid molecules and may be two or more orders ofmagnitude higher. In another embodiment, the slow off-rate enrichmentprocess is applied by diluting the sample mixture of nucleic acid-targetcomplexes several fold (e.g. at least about one of 2×, 3×, 4×, 5×) involume and incubating the mixture for some period of time beforepartitioning free from bound nucleic acids. The dilution volume isgenerally at least one order of magnitude higher, and may be about twoor more orders of magnitude higher, than the original volume. In yetanother embodiment, a combination of both competitor molecules anddilution is used to apply the slow off-rate enrichment process. Inanother embodiment, candidate mixtures that have been shown to result inan increased frequency of slow dissociation aptamers are used to selecta number of candidate aptamers. These aptamers are screened to identifyslow dissociation rate aptamers.

In another embodiment, a slow off-rate aptamer that includes a cleavableor releasable section in the fixed region of the aptamer is produced.The aptamer can also be produced with one or more of the followingadditional components: a labeled component, a spacer component, and aspecific binding tag. Any or all of these elements may be introducedinto a single stranded aptamer. In one embodiment, the element isintroduced at the 5′ end of the aptamer. In another embodiment, one ormore of these elements is included by creating a partially doublestranded aptamer, where one strand contains the various elements desiredas well as a sequence complementary to one of the fixed sequencesections of the second strand containing the variable target bindingregion.

A “releasable” or “cleavable” element or moiety or component refers to afunctional group where certain bonds in the functional group can bebroken to produce 2 separate components. In various embodiments, thefunctional group can be cleaved by irradiating the functional group(photocleavable) at the appropriate wavelength or by treatment with theappropriate chemical or enzymatic reagents. In another embodiment, thereleasable element may be a disulfide bond that can be treated with areducing agent to disrupt the bond. The releasable element allows anaptamer/target affinity complex that is attached to a solid support tobe separated from the solid support, such as by elution of the complex.The releasable element may be stable to the conditions of the rest ofthe assay and may be releasable under conditions that will not disruptthe aptamer/target complex.

As disclosed herein, an aptamer can further comprise a “tag” or“immobilization component or element” or “specific binding component orelement” which refers to a component that provides a means for attachingor immobilizing an aptamer (and any target molecule that is bound to it)to a solid support. A “tag” is a set of copies of one type or species ofcomponent that is capable of associating with a probe. “Tags” refers tomore than one such set of components. The tag can be attached to orincluded in the aptamer by any suitable method. Generally, the tagallows the aptamer to associate, either directly or indirectly, with aprobe or receptor that is attached to the solid support. The probe maybe highly specific in its interaction with the tag and retain thatassociation during all subsequent processing steps or procedures. A tagcan enable the localization of an aptamer affinity complex (or optionalcovalent aptamer affinity complex) to a spatially defined address on asolid support. Different tags, therefore, can enable the localization ofdifferent aptamer covalent complexes to different spatially definedaddresses on a solid support. A tag can be a polynucleotide, apolypeptide, a peptide nucleic acid, a locked nucleic acid, anoligosaccharide, a polysaccharide, an antibody, an affybody, an antibodymimic, a cell receptor, a ligand, a lipid, biotin, any fragment orderivative of these structures, any combination of the foregoing, or anyother structure with which a probe (or linker molecule, as describedbelow) can be designed or configured to bind or otherwise associate withspecificity. Generally, a tag is configured such that it does notinteract intramolecularly with either itself or the aptamer to which itis attached or of which it is a part. If SELEX is used to identify anaptamer, the tag may be added to the aptamer either pre- or post-SELEX.The tag is included on the 5′-end of the aptamer post-SELEX, or the tagis included on the 3′-end of the aptamer post-SELEX, or the tags may beincluded on both the 3′ and 5′ ends of the aptamers in a post-SELEXprocess. As illustrated in FIG. 9D, a fluorescent dye (such as Cy3), thephotocleavable and biotin moieties are all added to the end of theaptamer. Because of potential interactions between the photocleavablemoiety and the dye, a spacer is inserted between these two moieties. Allconstructs can be synthesized using standard phosphoramidite chemistry.Representative aptamer constructs are shown in FIG. 10A through FIG.10F. The functionality can be split between the 5′ and 3′ end orcombined on either end. In addition to photocleavable moieties, othercleavable moieties can be used, including chemically or enzymaticallycleavable moieties. A variety of spacer moieties can be used and one ormore biotin moieties can be included. Tags (also referred to asimmobilization or specific binding elements or components) other thanbiotin can also be incorporated. Suitable construction reagents includebiotin phosphoramidite, PC Linker (Glen Research PN 10-4920-02); PCbiotin phosphoramidite (Glen Research PN 10-4950-02); dSpacer CEphosphoramidite (Glen Research PN 10-1914-02); Cy3 phosphoramidite (GlenResearch PN 10-5913-02); and Arm26-Ach Spacer Amidite (Fidelity SystemsPN SP26Ach-05). This type of tag on a target specific slow off-rateaptamer may be used to introduce secondary reagents, such as a label,into a tissue or cell sample. For example if the slow off-rate aptamercontains a biotin tag, a labeled avidin molecule could be introduced togenerate signal.

In one embodiment, base modifications of the nucleotides are used in theproduction of the variable region of the aptamer. These modifiednucleotides have been shown to produce aptamers that have very slowoff-rates from their targets. In the methods of the present disclosurethe candidate mixture may comprise modified nucleic acids in which one,several (e.g. one of, or at least one of, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30) or all pyrimidines in at least one, or each, nucleic acid of thecandidate mixture is chemically modified at the 5-position. Optionally,all C residues in the nucleic acids of the candidate mixture arechemically modified at the 5-position. Optionally, all T residues in thenucleic acids of the candidate mixture are chemically modified at the5-position. Optionally, all U residues in the nucleic acids of thecandidate mixture are chemically modified at the 5-position.

A “cytological specimen or sample” may include a wide range of specimentypes. These include abdominal and pelvic washings, body cavity fluids(pleural, peritoneal), urine, gastric/esophageal washings, fine needleaspirates (FNA), breast fluid, CSF, cyst fluid, synovial fluid, andbronchial washings. Smears may be prepared from FNA specimens or brushcollected specimens as is done for PAP Smears.

A “cytology protocol” generally consists of sample collection, samplefixation, sample immobilization, and staining. “Cell preparation” mayinclude all of the processing steps after sample collection includingthe use of one or more slow off-rate aptamer for the staining of theprepared cells.

Sample collection may involve directly placing the sample in anuntreated transport container, placing the sample in a transportcontainer containing some type of media, or placing the sample directlyonto a slide (immobilization) without any treatment or fixation.

Sample immobilization may be improved by applying a portion of thecollected specimen to a glass slide that is treated with polylysine,gelatin, or a silane. Slides may be prepared by smearing a thin and evenlayer of cells across the slide. Care is taken to minimize mechanicaldistortion and drying artifacts. Liquid specimens may be processed in acell block method or liquid specimens may be mixed 1:1 with the fixativesolution for 10 minutes at room temperature.

Cell blocks may be prepared from residual effusions, sputum, urinesediments, gastrointestinal fluids, cell scraping, or fine needleaspirates. Cells are concentrated or packed by centrifugation ormembrane filtration. A number of methods for cell block preparation havebeen developed. Representative procedures include the fixed sediment,bacterial agar, or membrane filtration methods. In the fixed sedimentmethod, the cell sediment is mixed with a fixative like Bouin's, picricacid, or buffered formalin and then the mixture is centrifuged to pelletthe fixed cells. The supernatant is removed, drying the cell pellet ascompletely as possible. The pellet is collected and wrapped in lenspaper and then placed in a tissue cassette. The tissue cassette isplaced in a jar with additional fixative and processed as a tissuesample. Agar method is very similar but the pellet is removed and driedon paper towel and then cut in half. The cut side is placed in a drop ofmelted agar on a glass slide and then the pellet is covered with agarmaking sure that no bubbles form in the agar. The agar is allowed toharden and then any excess agar is trimmed away. This is placed in atissue cassette and the tissue process completed. Alternatively, thepellet may be directly suspended in 2% liquid agar at 65° C. and thesample centrifuged. The agar cell pellet is allowed to solidify for anhour at 4° C. The solid agar may be removed from the centrifuge tube andsliced in half. The agar is wrapped in filter paper and then the tissuecassette. Processing from this step on is as above. The centrifugationsteps may be replaced in any these procedures with a membrane filtrationstep. Any of these processes may be used to generate a “cell blocksample”.

Cell blocks may be prepared using specialized resins including Lowicrylresins, LR White, LR Gold, Unicryl, and MonoStep. These resins have lowviscosity and can be polymerized, at low temperatures and ultra violet(UV) light. The embedding process relies on progressively cooling thesample during the dehydration steps, transferring the sample to theresin and polymerizing a block at the final low temperature at theappropriate UV wavelength.

Cell block sections may be stained with hematoxylin-eosin forcytomorphological examination while additional sections are used forexamination for specific markers.

Whether the process is cytological or histological, the sample may befixed prior to additional processing to prevent sample degradation. Thisstep is called “fixation” and describes a wide range of materials andprocedures that may be used interchangeably. The sample fixationprotocol and reagents are best selected empirically based on the targetsto be detected and the specific cell/tissue type to be analyzed. Samplefixation relies on reagents such as ethanol, polyethylene glycol,methanol, formalin, or isopropanol. The samples should be fixed as soonafter collection and affixation to the slide as possible. However, thefixative selected can introduce structural changes into variousmolecular targets making their subsequent detection more difficult. Thefixation and immobilization processes and their sequence can modify theappearance of the cell and these changes must be anticipated andrecognized by the cytotechnologist. Fixatives can cause shrinkage ofcertain cell types and cause the cytoplasm to appear granular orreticular. Many fixatives function by crosslinking cellular components.This can damage or modify specific epitopes, generate new epitopes,cause molecular associations, and reduce membrane permeability. Formalinfixation is one of the most common cytological/histological approaches.Formalin forms methyl bridges between neighboring proteins or withinproteins. Precipitation or coagulation is also used for fixation andethanol is frequently used in this type of fixation. A combination ofcrosslinking and precipitation can also be used for fixation. A strongfixation process is best at preserving morphological information while aweaker fixation process is best the preservation of molecular targets.

A representative fixative would be 50% absolute ethanol, 2 mMpolyethylene glycol (PEG), 1.85% formaldehyde. Variations on thisformulation include ethanol (50% to 95%), methanol (20%-50%), andformalin (formaldehyde) only. Another common fixative is 2% PEG 1500,50% ethanol, and 3% methanol. Slides are place in the fixative for 10 to15 minutes at room temperature and then removed and allowed to dry. Onceslides are fixed they can be rinsed with a buffered solution like PBS.

A wide range of dyes can be used to differentially highlight andcontract or “stain” cellular, sub-cellular, and tissue features ormorphological structures. Hematoylin is used to stain nuclei a blue orblack color. Orange G-6 and Eosin Azure both stain the cell's cytoplasm.Orange G stains keratin and glycogen containing cells yellow. Eosin Y isused to stain nucleoli, cilia, red blood cells, and superficialepithelial squamous cells. Romanowsky stains are used for air driedslides and are useful in enhancing pleomorphism and distinguishingextracellular from intra-cytoplasmic material.

The staining process may involve a treatment to increase thepermeability of the cells to the stain. Treatment of the cells with adetergent may be used to increase permeability. To increase cell andtissue permeability fixed samples may be further treated with solvents,saponins, or non-ionic detergents. Enzymatic digestion may also improvethe accessibility of specific targets in a tissue sample.

After staining the sample is dehydrated using a succession of alcoholrinses with increasing alcohol concentration. The final wash is donewith xylene or a xylene substitute, such as a citrus terpene, that has arefractive index close to that of the coverslip to be applied to theslide. This final step is referred to as clearing. Once the sample isdehydrated and cleared a mounting medium is applied. The mounting mediumis selected to have a refractive index close to the glass and is capableof bonding the coverslip to the slide. It will also prevent theadditional drying, shrinking, or fading of the cell sample.

Regardless of the stains or processing steps used the final evaluationof the cytological specimen is made by some type of microscopy and avisual inspection of the morphology and determination of the markerpresence or absence. Microscopic methods utilized include brightfield,phase contract, fluorescence, and differential interference constrast.

If secondary tests are required on the sample after examination, thecoverslip may be removed and the slide destained. Destaining involvesusing the original solvent systems used in staining the slide originallywithout the added dye and in a reverse order to the original stainingprocedure. Destaining may also be completed by soaking the slide in anacid alcohol until the cells are colorless. Once colorless the slidesare rinsed well in a water bath and the second staining procedureapplied.

In addition, specific molecular differentiation may be possible inconjunction with the cellular morphological analysis through the use ofspecific molecular reagents such as antibodies or nucleic acid probes.This improves the accuracy of diagnostic cytology. Micro-dissection maybe used to isolate a subset of cells for additional evaluation, inparticular, for genetic evaluation of abnormal chromosomes, geneexpression, or mutations.

In histology, tissue specimens may be collected from epithelium,connective tissues, cartilage, bone, muscle, nerves, vessels, heart,lymphatic system, respiratory tract, urinary tract, endocrine system,and reproductive system.

Preparation of a tissue sample for histological evaluation involvesfixation, dehydration, infiltration, embedding, and sectioning. Thefixation reagents used in histology are very similar or identical tothose used in cytology and have the same issues of preservingmorphological features at the expense of molecular ones such asindividual proteins. Time can be saved if the tissue sample is not fixedand dehydrated but instead is frozen and then sectioned while frozen.This is a more gentle processing step and can preserve more individualmarkers. However, freezing is not acceptable for long term storage of atissue sample as subcellular information is lost due to the introductionof ice crystals. Ice in the frozen tissue sample also prevents thesectioning process from producing a very thin slice and thus somemicroscopic resolution and imaging of subcellular structures can belost. In addition to formalin fixation, osmium tetroxide is used to fixand stain phospholipids (membranes).

Dehydration of tissues is accomplished with successive washes ofincreasing alcohol concentration. Clearing requires a material that ismiscible with alcohol and the embedding material and involves a stepwiseprocess starting at 50:50 alcohol:clearing reagent and then 100%clearing agent (xylene or xylene substitute). Infiltration involvesincubating the tissue with a liquid form of the embedding agent (warmwax, nitrocellulose solution) first at 50:50 embedding agent: clearingagent and the 100% embedding agent. Embedding is completed by placingthe tissue in a mold or cassette and filling with melted embedding agentsuch as wax, agar, or gelatin. The embedding agent is allowed to harden.The harden tissue sample may then be sliced into thin section forstaining and subsequent examination.

Prior to staining, the tissue section is dewaxed and rehydrated. Xyleneis used to dewax the section, one or more changes of xylene may be used,and the tissue is rehydrated by successive washes in alcohol ofdecreasing concentration. Prior to dewax the tissue section may be heatimmobilized to a glass slide at 80° C. for 20 minutes. Histology stainsare listed in FIG. 1.

Laser capture micro-dissection allows the isolation of a subset of cellsfor further analysis from a tissue section.

As in cytology, to enhance the visualization of the microscopicfeatures, the tissue section or slice may be stained with a variety ofstains. A large menu of stains are available to enhance or identifyspecific features.

To further increase the interaction of immunological reagents withcytological/histological samples a number of techniques for “antigenretrieval” have been developed. The first such technique relied on hightemperature heating of a fixed sample. This method is also referred toas heat-induced epitope retrieval or HIER. A variety of heatingtechniques have been used, including steam heating, microwaving,autoclaving, water baths, and pressure cooking or a combination of thesemethods of heating. Antigen retrieval solutions include water, citrate,or normal saline buffers. The key to antigen retrieval is the time athigh temperature but lower temperatures for longer times have also beensuccessfully used. Another key to antigen retrieval is the pH of theheating solution. Low pH was found to provide the best immunostainingbut also gave rise to backgrounds that required the use of a secondtissue section as a negative control. The most consistent benefit(increased immunostaining without increase in background) was found witha high pH solution regardless of the buffer composition. The antigenretrieval process for a specific target should be empirically optimizedfor that target using heat, time, pH, and buffer composition asvariables for process optimization. Using the microwave antigenretrieval method has allowed for sequential staining of differenttargets with antibody reagents. But the time required to antibody andenzyme complexes between staining steps has also been shown to degradecell membrane antigens. Microwave heating methods have improved in situhybridization methods as well.

To initiate the antigen retrieval process, the section is first dewaxedand hydrated. The slide is then placed in 10 mM sodium citrate buffer pH6.0 in a dish or jar. A representative procedure uses an 1100 Wmicrowave and microwaves the slide at 100% power for 2 minutes followedby microwaving the slides using 20% power for 18 minutes after checkingto be sure the slide remains covered in liquid. The slide is thenallowed to cool in the uncovered container and then rinsed withdistilled water. HIER may be used in combination with an enzymaticdigestion to improve the reactivity of the target to immunochemicalreagents.

One such enzymatic digestion protocol uses proteinase K. A 20 μg/mlconcentration of proteinase K is prepared in 5 0 mM Tris Base, 1 mMEDTA, 0.5% Triton X-100, pH 8.0 buffer. The process first involvesdewaxing sections in 2 changes of xylene, 5 minutes each. Then thesample is hydrated in 2 changes of 100% ethanol for 3 minutes each, 95%and 80% ethanol for 1 minute each and then rinsed in distilled water.Sections are covered with Proteinase K working solution and incubated10-20 minutes at 37° C. in humidified chamber (optimal incubation timemay vary depending on tissue type and degree of fixation). The sectionsare cooled at room temperature for 10 minutes and then rinsed in PBSTween 20 for 2×2 min. If desired sections may be blocked to eliminatepotential interference from endogenous compounds and enzymes. Thesection is then incubated with primary antibody at appropriate dilutionin primary antibody dilution buffer for 1 hour at room temperature orovernight at 4° C. The section is then rinsed with PBS Tween 20 for 2×2min. Additional blocking may be performed if required for the specificapplication followed by additional rinsing with PBS Tween 20 for 3×2 minand then finally the immunostaining protocol completed.

A simple treatment with 1% SDS at room temperature has also beendemonstrated to improve immunohistochemical staining. Antigen retrievalmethods have been applied to slide mounted sections as well as freefloating sections. Another treatment option is to place the slide in ajar containing citric acid and 0.1 Nonident P40 at pH 6.0 and heating to95° C. The slide is then washed with a buffer solution like PBS.

For immunological staining of tissues it may be useful to blocknon-specific association of the antibody with tissue proteins by soakingthe section in a protein solution like serum or non-fat dry milk.

An “slow off-rate aptamer treated sample” is intended to mean anycytological or histological slide or section that may be treated withone or more slow off-rate aptamers to one or more targets to bedetected. The treatment process may include placing the slide or sectionin one or more buffer or reagent at one or more temperatures for aperiod of time sufficient to complete the desired interaction betweenthe slow off-rate aptamer and target, the slow off-rate aptamer andsubsequent detection moiety, partitioning, blocking reactions, or otherprocess steps.

Blocking reactions may include the need to reduce the level ofendogenous biotin; eliminate endogenous charge effects; inactivateendogenous nucleases; and/or inactivate endogenous enzymes likeperoxidase and alkaline phosphatase. Endogenous nucleases may beinactivated by degradation with proteinase K, by heat treatment, use ofa chelating agent such as EDTA or EGTA, the introduction of carrier DNAor RNA, treatment with a chaotrope such as urea, thiourea, guanidinehydrochloride, guanidine thiocyanate, lithium perchlorate, etc, ordiethyl pyrocarbonate. Alkaline phosphatase may be inactivated bytreated with 0.1N HCl for 5 minutes at room temperature or treatmentwith 1 mM levamisole. Peroxidase activity may be eliminated by treatmentwith 0.03% hydrogen peroxide. Endogenous biotin may be blocked bysoaking the slide or section in an avidin (streptavidin, neutravidin maybe substituted) solution for at least 15 minutes at room temperature.The slide or section is then washed for at least 10 minutes in buffer.This may be repeated at least three times. Then the slide or section issoaked in a biotin solution for 10 minutes. This may be repeated atleast three times with a fresh biotin solution each time. The bufferwash procedure is repeated. All slides or sections used for a singlediagnostic purposes should be treated with the same blocking protocol.Blocking protocols should be minimized to prevent damaging either thecell or tissue structure or the target or targets of interest but one ormore of these protocols could be combined to “block” a slide or sectionprior to reaction with one or more slow off-rate aptamers.

In one embodiment, a cytological sample may be collected, applied to aglass slide, fixed, stained for the structural/morphological featuresappropriate to the sample collected and disease state to be diagnosed,microscopically examined, and then may be reacted with one or slowoff-rate aptamers to the desired target or targets. The method mayfurther include one or more of the following steps; an antigen retrievalstep; treating to increase the cell permeability (permeabilizing); oneor more blocking step; dehydrating; clearing; wash steps; and/or adestaining step. The sequence of the individual steps may beinterchanged as needed. The slow off-rate aptamers may optionally bedesigned to crosslink with their specific target.

In one embodiment, a cytological sample may be collected, applied to aglass slide, fixed, stained for the structural/morphological featuresappropriate to the sample collected and disease state to be diagnosed,microscopically examined, and then may be reacted with one orcrosslinking slow off-rate aptamers or photoaptamers to the desiredtarget or targets. The method may further include one or more of thefollowing steps; an antigen retrieval step; treating to increase thecell permeability (permeabilizing); dehydrating; clearing; one or moreblocking step; wash steps; and/or a destaining step. The sequence of theindividual steps may be interchanged as needed but includes anadditional step to activate the crosslinking process.

In another embodiment sections from a cell block procedure may beutilized. When a cell block is used the sample may be prepared as if itwere a tissue sample. One section may be stained as is appropriate tothe cell type and disease state to be diagnosed. Another section may betreated with the one or more slow off-rate aptamers for detection ofspecific target or targets. And another section may be treated with thesame reagents and sequence as the slow off-rate aptamer treated sectionin the absence of the one or more slow off-rate aptamers to serve as anegative control section. Optionally these slow off-rate aptamers may becrosslinking slow off-rate aptamers or photoaptamers. These proceduresmay be applied to tissue sections.

In some embodiments, once the slow off-rate aptamer(s) is allowed toequilibrate with the tissue or cell sample to form an slow off-rateaptamer target affinity complex, a kinetic challenge may be used. If akinetic challenge is introduced, non-specific complexes between the slowoff-rate aptamer and any non-target molecules are unlikely to re-formfollowing their dissociation. Since non-specific complexes generallydissociate more rapidly than an slow off-rate aptamer affinity complex,a kinetic challenge reduces the likelihood that an slow off-rate aptamerwill be involved in a non-specific complex with a non-target. Aneffective kinetic challenge can provide the assay with additionalspecificity, beyond that of the initial slow off-rate aptamer bindingevent and any subsequent optional covalent interaction. Thus, thekinetic challenge offers a second determinant of specificity. In oneembodiment, 10 mM dextran sulfate is added to the slow off-rate aptameraffinity complexes that are tissue or cell associated, and is incubatedfor about 15 minutes. In another embodiment, the kinetic challenge isinitiated in the presence of 10 mM dextran sulfate. In the case of akinetic challenge that uses a competitor, the competitor can also be anymolecule that can form a non-specific complex with a free slow off-rateaptamer, for example to prevent that slow off-rate aptamer fromrebinding non-specifically to a non-target molecule. Such competitormolecules include polycations (e.g., spermine, spermidine, polylysine,and polyarginine) and amino acids (e.g., arginine and lysine). When acompetitor is used as the kinetic challenge a fairly high concentrationis utilized relative to the anticipated concentration of total proteinor total slow off-rate aptamer present in the sample. In one embodiment,about 10 mM dextran sulfate is used as the competitor in a kineticchallenge. In one embodiment, the kinetic challenge comprises adding acompetitor to the tissue or cell sample containing the slow off-rateaptamer affinity complex, and incubating the sample for a time ofgreater than or equal to about 30 seconds, about 1 minute, about 2minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10minutes, about 30 minutes, and about 60 minutes. In another embodiment,the kinetic challenge comprises adding a competitor to the tissue orcell sample containing the slow off-rate aptamer affinity complex andincubating for a time such that the ratio of the measured level of slowoff-rate aptamer affinity complex to the measured level of thenon-specific complex is increased.

In some embodiments, the kinetic challenge is performed by contactingthe test sample with binding buffer or any other solution that does notsignificantly increase the natural rate of dissociation of slow off-rateaptamer affinity complexes. The dilution can be about 2×, about 3×,about 4×, about 5×, or any suitable greater dilution. Larger dilutionsprovide a more effective kinetic challenge by reducing the concentrationof total protein and slow off-rate aptamer after dilution and,therefore, the rate of their re-association. In one embodiment, the slowoff-rate aptamer affinity complex is effectively diluted by addition ofthe diluent and incubated for a time ≧about 30 seconds, ≧about 1 minute,≧about 2 minutes, ≧about 3 minutes, ≧about 4 minutes, ≧about 5 minutes,≧about 10 minutes, ≧about 30 minutes, and ≧about 60 minutes. In anotherembodiment, the slow off-rate aptamer affinity complex is effectivelydiluted by addition of a diluent and incubated for a time such that theratio of the measured level of slow off-rate aptamer affinity complex tothe measured level of the non-specific complex is increased.

In some embodiments, the kinetic challenge is performed in such a mannerthat the effect of sample dilution and the effect of introducing acompetitor are realized simultaneously. For example, a tissue or cellsample can be effectively diluted by addition of a large volume ofcompetitor. Combining these two kinetic challenge strategies may providea more effective kinetic challenge than can be achieved using onestrategy. In one embodiment, the effective dilution can be about 2×,about 3×, about 4×, about 5×, or any suitable greater dilution and thecompetitor is about 10 mM dextran sulfate. In one embodiment, thekinetic challenge comprises contacting the tissue or cell samplecontaining the slow off-rate aptamer affinity complex with a volume ofdiluent, adding a competitor to the mixture containing the slow off-rateaptamer affinity complex, and incubating the mixture containing the slowoff-rate aptamer affinity complex for a time greater than or equal toabout 30 seconds, about 1 minute, about 2 minutes, about 3 minutes,about 4 minutes, about 5 minutes, about 10 minutes, about 30 minutes,and about 60 minutes. In another embodiment, the kinetic challengecomprises diluting the mixture containing the slow off-rate aptameraffinity complex, adding a competitor to the mixture containing the slowoff-rate aptamer affinity complex and incubating the mixture containingthe slow off-rate aptamer affinity complex for a time such that theratio of the measured level of slow off-rate aptamer affinity complex tothe measured level of the non-specific complex is increased. In oneembodiment, the crosslinking step is introduced into the procedurefollowing the kinetic challenge step. In another embodiment, thecrosslinking step is introduced into the procedure following a kineticchallenge step and a wash step.

In another embodiment a tissue specimen is collected, frozen or fixed,dehydrated, infiltrated with an embedding media, embedded in theembedding media, sliced, mounted, cleared, stained for the appropriatefeatures for the sample collected and the disease state to be diagnosed,microscopically examined, and then reacted with one or more slowoff-rate aptamers to the desired target or targets. The method mayfurther include one or more of the following steps; an antigen retrievalstep; treating to increase the tissue permeability (permeabilizing); oneor more blocking step; dehydrating; wash steps; and/or a destainingstep. The sequence of the individual steps may be interchanged asneeded. All processes up to the first microscopic examination may becompleted on free floating tissue section. Optionally these slowoff-rate aptamers may be crosslinking slow off-rate aptamers orphotoaptamers.

In another embodiment multiple tissue sections are used in thediagnostic procedure. One section is stained as is appropriate to thetissue type and disease state to be diagnosed for evaluation of themorphological structures and features of the tissue. Another section istreated with the one or more slow off-rate aptamers for detection ofspecific target or targets in a buffered solution. And another sectionis treated with the same reagents and sequence as the slow off-rateaptamer treated section in the absence of the one or more slow off-rateaptamers to serve as a negative control section where the slow off-rateaptamer reaction step is replaced by treatment with the bufferedsolution used in the slow off-rate aptamer reaction step.

In another embodiment the slide mounted smear, cell block, or tissuesection may be treated to dewax the sample and then may be rehydratedprior to staining. The stain selected will be appropriate to the sampleand disease state to be diagnosed. Prior to microscopic evaluation, theslide mounted sample may be covered with a coverslip. When themicroscopic evaluation of the morphological or structural features iscomplete, then the coverslip may be removed and the sample may beoptionally treated with a reagent, such as acid alcohol, to destain thesample. If a destaining protocol is used the sample should be washedseveral times with deionized water. To prepare the sample for reactionwith one or more target specific slow off-rate aptamers, a blockingprotocol may optionally be used. One embodiment uses dextran sulfate(DexSO₄) or another polyanionic compound as a blocking agent. Otherpolyanionic materials could include heparin, herring sperm DNA, salmonsperm DNA, tRNA, polydextran, abasic phosphodiester polymers, dNTPs, andpyrophosphate. In one embodiment the slow off-rate aptamer rinsesolution may contain a polyanionic material like dextran sulfate. Abuffered solution of one or more slow off-rate aptamers at aconcentration of 1-20 nM of each slow off-rate aptamer may be used. Theslow off-rate aptamer solution may be applied to the sample andincubated at room temperature or 37° C. for a period of time selected(empirically) to maximize reaction, or binding, with the specific targetor targets of interest. Incubation times may be as long as 18 hours. Thebuffered slow off-rate aptamer solution may contain a variety of othermaterials such as a non-specific polynucleotide to minimize non-specificinteraction of the slow off-rate aptamer/s with nucleic acid bindingsites in or on the tissue or cell sample. The buffered slow off-rateaptamer solution may then be rinsed from the sample using an slowoff-rate aptamer rinse solution. The coverslip may be reapplied and thesample examined microscopically for the detection one or more specificdetectable moiety that may be introduced to the sample through the slowoff-rate aptamer.

In another embodiment, the detectable moiety introduced to the samplethrough the binding of the slow off-rate aptamer may be a fluorescent,chemilluminescent, or colorimetric detectable moiety that is directlyattached to the slow off-rate aptamer. When more than one slow off-rateaptamer is used in the buffered slow off-rate aptamer solution, eachslow off-rate aptamer may include a detectable moiety with an uniquewavelength of detection and/or excitation. Thus multiple targets may bedetected simultaneously. Optionally these slow off-rate aptamers may bedesigned to crosslink to their specific target.

In other embodiments, the targets may be detected sequentially. In thisembodiment, once the morphological evaluation with a traditional stainis complete, the sample may be reacted with a first slow off-rateaptamer to a specific first target and the first detectable moiety maybe introduced by the presence of the first slow off-rate aptamer if thespecific first target is present in the sample. Then the sample may betreated with conditions of heat, buffer, pH, ionic strength sufficientto cause the first slow off-rate aptamer/first target pair todissociate. The first slow off-rate aptamer may then be washed from thesample and a second slow off-rate aptamer specific to a second targetreacted with the sample. The second detectable moiety may then bedetected if the second target is present in the sample. This cycle maybe completed until the all desired targets have been evaluated.

In another embodiment, the slow off-rate aptamer or slow off-rateaptamers may be designed with an element to support signal generation.In one embodiment, the element to support signal generation may be anenzyme attached to the slow off-rate aptamer, or attached via a tag,such that it does not interfere with binding of the slow off-rateaptamer to the target. Once the slow off-rate aptamer has bound to thespecific target and excess slow off-rate aptamer, or slow off-rateaptamers, has been removed, the enzyme may be reacted with its specificsubstrate to produce a detectable signal at the site where the enzymemay be immobilized. A precipitating, calorimetric or fluorescentsubstrate may be used. In another embodiment, the enzyme attached to theslow off-rate aptamer may be used to increase the signal. In anotherembodiment, the element to support signal generation consists of twocomponents. The first component of the element to support signalgeneration is designed into the slow off-rate aptamer and is ligand likebiotin that reacts with a corresponding receptor like avidin, the secondcomponent of the element to support signal generation. The secondcomponent may be attached to the detectable moiety.

In another embodiment, the slow off-rate aptamer reacted with a specifictarget in the sample may be used in combination or serve as the nucleicacid target in a variety of nucleic acid amplification methods includingPCR, rolling circle amplification, q-beta replicase, stranddisplacement, helicase dependent amplification, loop mediated isothermalamplification, ligase chain reaction, restriction and circularizationaided rolling circle amplification, etc. For example, the targetimmobilized slow off-rate aptamer may serve as a template for a PCRreaction to produce multiple copies of a PCR product in a solutioncovering the sample. Detection of the PCR product could be completed awide variety of methods by hybridization of a labeled PCR product to anarray, by real time PCR measurement, by gel electrophoresis, sequencingmethods, etc.

In another embodiment, the tissue or cell sample is reacted with one ormore slow off-rate aptamers prior to any morphological stainingprocedure.

One embodiment for the use of slow off-rate aptamers in a cytologicalevaluation would be the combination of a PAP smear evaluation with slowoff-rate aptamers specific to molecular targets from the high risk HPV16and/or 18 strains. In this case the slow off-rate aptamers may bedesigned to react with E6 or E7 proteins or to E6 or E7 and one of L1 orE2 or L2 proteins. A representative PAP smear staining protocol utilizesa combination of dyes, Harris hematoxylin, orange G6, and EA 50. EA 50is a combination dye that contains eosin Y, Bismarck brown, and fastgreen. Hematoxylin stains nuclei blue, the other dyes react with keratinto stain the cell cytoplasm from green to blue or pink depending on thekeratin content. The staining protocol may be started with thehematoxylin (5 minutes). Each staining step may be followed with anumber of washes (for example 3) before the next step. Washing cyclesmay be eliminated after the alcohol treatment step. The slide may thenbe treated with 0.1% HCl solution for 1 minute, then with 0.02% ammoniumwater for 1 minute, 95% reagent grade ethanol for 2 minutes for 2cycles, Orange G6 for 2 minutes, 95% reagent grade ethanol for 2 minutesand 2 cycles, EA 50 for 3 minutes, and finally 95% reagent grade ethanolfor 2 minutes and 2 cycles. The slides may be dehydrated with alcoholand cleared with xylene and then a mounting medium applied and acoverslip may be applied. Slides may then be examined microscopically.The coverslip may then be removed and the slide may be treated with oneor more rinses of acid alcohol to remove the dyes used for themorphological examination. Once the smear is destained, the slide couldbe covered with a reaction solution containing the slow off-rate aptamer(or slow off-rate aptamers) and incubated from 30 minutes to overnight.The incubation period should be selected to optimize slow off-rateaptamer association with its specific target while minimizing backgroundstaining.

A reaction or buffered solution could independently contain 0.005-40 nMof one or more slow off-rate aptamers each specific to a target ofinterest. For example, the slow off-rate aptamer concentration could be≦ any of the following concentrations; 0.005 nM, 1 nM, 2 nM, 4 nM, 8 nM,16 nM, 32 nM, 35 nM, or 40 nM by slow off-rate aptamer in the mix. Thereaction solution could contain a buffer such SB17 (40 mM HEPES, pH 7.5,125 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 0.05% TWEEN-20) or otherbuffer selected empirically to minimize the changes in cells or tissuesor promote slow off-rate aptamer diffusion into the section (tissue orcell). The reaction solution may further contain materials to minimizenon-specific binding of the slow off-rate aptamer to sample derivednucleic acids including for example herring sperm DNA, etc. Additionalcomponents could include dextran sulfate, a carrier protein like casein,etc. An optional kinetic challenge step could be utilized.

In one embodiment, tissue sections or cells from a number of relatedtumor types may be used to select and produce slow off-rate aptamersthat react with a desired tumor marker. Because the marker in thedifferent tissue sections may be crosslinked or associated withdifferent materials in the different tissue sections it may be possibleto select and produce slow off-rate aptamers that differentiate thepresence of that particular marker in the specific, localizedenvironment that is unique to that tumor type. Thus it may be possibleto identify a panel of slow off-rate aptamers that can be used todifferentiate, for example, anaplastic oligodendrogliom, astrocytomas,and oligoastrocytomas. Optionally these slow off-rate aptamers may bephotoaptamers or crosslinking aptamers.

In another embodiment, the slow off-rate aptamers selected and producedto differentiate tumor types, may be produced such that each slowoff-rate aptamer in a reaction mixture contains an unique fluorescent orother type of label that would produce a specific signal unique to thatslow off-rate aptamer. Detection of an unique combination of labelswould be used to provide the differentiation of the type of tumor. Eachslow off-rate aptamer could also be produced with multiple copies of thefluorescent or other label to increase signal generated upon interactionof the slow off-rate aptamer and its specific target in a tissue sectionor cell preparation. Signal generating labels may be selected to beclearly visible in stained cells or tissues eliminating the need todestain the sample before reaction with the slow off-rate aptamer orslow off-rate aptamers. Optionally these slow off-rate aptamers may bephotoaptamers or crosslinking aptamers.

In another embodiment, the tissue section or cells may be reacted withslow off-rate aptamers selected and produced to a specific tumor marker.In addition to the slow off-rate aptamer or slow off-rate aptamers tothe tumor marker, the tissue section or cells may be reacted with one ormore slow off-rate aptamers that are specific to one or more hormone orother type of tumor specific marker that may be produced by the tumorand help identify the tumor type and origin.

In another embodiment, the tissue section or cells may be reacted withone or more slow off-rate aptamer selected and produced to one or moremarker that may be used to establish the prognosis for the disease. Inone embodiment, the tissue section or cells may be reacted with one ormore slow off-rate aptamer selected and produced to one or more markerthat may be used to support selection of appropriate therapeutic agents.For example, the presence of a high level of a specific glycosylhydrolase, YKL-40, in a glioma may indicate a poorer prognosis than in aglioma that has less YKL-40. Differential expression of the YKL-40 levelmay be established by the level of slow off-rate aptamer stainingobserved in the tissue section or cell preparation relative to a controlmaterial.

In any of the embodiments for the analysis of a tissue or cell sample,after initial incubation of the one or more slow-off rate aptamers withthe sample, the slow off-rate aptamer or slow off-rate aptamers may beoptionally crosslinked to their corresponding targets by exposure to theappropriate crosslinking activator.

In another embodiment, a histological or cytological reagent is providedthat may consist of one or more slow off-rate aptamers specific to oneor more targets that are indicative of a specific disease state. Targetsmay include tumor specific markers, hormones, or other molecules. Inaddition to the slow off-rate aptamers, the reagent may consist ofbuffers, salts, detergents, blocking reagents, competitors, andstabilizers.

The method of the instant disclosure is illustrated generally inExamples 1-8. Example 1 describes the general affinity SELEX methodusing a candidate mixture comprised of modified nucleotides. Example 2describes a photo SELEX method using a candidate mixture comprised ofmodified nucleotides and a 5′-terminal photoreactive group, and theimproved SELEX method in which dilution is used to provide the slowoff-rate enrichment process to the equilibrated aptamer:target mixture.Example 3 extends the method described in Example 2 by the addition of acompetitor to the dilution step. Example 4 illustrates the effectivenessof the slow off-rate enrichment process. The average dissociationhalf-life value (t_(1/2)) for aptamers using the modified nucleotides5-benzyl-dUTP (BzdUTP), 5-isobutyl-dUTP (iBdUTP), or 5-tryptamino-dUTPselected in the absence of a slow off-rate enrichment process was 20minutes with some aptamers having a t_(1/2) value of up to one hour.This is substantially longer than what has been previously describedwith natural bases or other modified nucleotides. The average foraptamers selected with a slow off-rate enrichment process was over 85minutes. More specifically, with reference to FIG. 4B, it can be seenthat introduction of a slow off-rate enrichment process producedaptamers with t_(1/2) values of about ≧30 min., ≧about 60 min., ≧about90 min., ≧about 120 min., ≧about 150 min., ≧about 180 min., ≧about 210min., and ≧about 240 min. These dissociation rates for aptamer:targetcomplexes are unprecedented.

Example 5 describes the generation of slow off-rate aptamers using aNpdUTP (napthyl-dUTP) candidate mixture.

Example 6 describes the generation of a slow off-rate aptamer to apeptide target.

Example 7 illustrates the utility of slow off-rate aptamers relative toconventional aptamers.

Example 8 illustrates the generation of slow off-rate aptamers using aBZdU candidate mixture.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention as defined in theappended claims.

Example 1 Incorporation of Modified Nucleotides in Nucleic AcidLibraries Leads to Higher Affinity Enriched Libraries in Affinity SELEX

A. Preparation of Candidate Mixtures

Candidate mixtures were prepared with dATP, dGTP, 5-methyl-dCTP (MedCTP)and either dTTP or one of three dUTP analogs: 5-Benzyl-dUTP (BzdUTP),5-isobutyl-dUTP (iBdUTP), or 5-tryptamino-dUTP (TrpdUTP). Candidatemixtures were prepared by polymerase extension of a primer annealed to abiotinylated template (FIG. 3). For each candidate mixture composition,4.8 nmol forward PCR primer and 4 nmol template were combined in 100 μL1×KOD DNA Polymerase Buffer (Novagen), heated to 95° C. for 8 minutes,and cooled on ice. Each 100 μL primer:template mixture was added to a400 μL extension reaction containing 1×KOD DNA Polymerase Buffer, 0.125U/μL KOD XL DNA Polymerase, and 0.5 mM each dATP, MedCTP, dGTP, and dTTPor dUTP analog, and incubated at 70° C. for 30 minutes. Double-strandedproduct was captured via the template strand biotins by adding 1 mLstreptavidin-coated magnetic beads (MagnaBind Streptavidin, Pierce, 5mg/mL in 1M NaCl+0.05% TWEEN-20) and incubating at 25° C. for 10 minuteswith mixing. Beads were washed three times with 0.75 mL SB1T Buffer (40mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KC1, 1 mM MgCl₂, 1 mM CaCl₂, 0.05%TWEEN-20). The aptamer strand was eluted from the beads with 1.2 mL 20mM NaOH, neutralized with 0.3 mL 80 mM HCl, and buffered with 15 μL 1 MHEPES, pH 7.5. Candidate mixtures were concentrated with a Centricon-30to approximately 0.2 mL, and quantified by UV absorbance spectroscopy.

B. Immobilization of Target Proteins

Target proteins were purchased with poly His tags, such as, (His)₆ tags(R&D Systems) and immobilized on Co⁺²-NTA paramagnetic beads (MyOneTALON, Invitrogen, or hereinafter referred to as Talon beads). Targetproteins were diluted to 0.2 mg/mL in 0.5 mL BIW Buffer (50 mMNa-phosphate, pH 8.0, 300 mM NaCl, 0.01% TWEEN-20), and added to 0.5 mLTALON beads (pre-washed three times with B/W Buffer and resuspended to10 mg/mL in B/W Buffer). The mixture was rotated for 30 minutes at 25°C. and stored at 4° C. until use. TALON beads coated with (His)₆ peptidewere also prepared and stored as above. Prior to use, beads were washed3 times with B/W Buffer, once with SB1T, and resuspended in SB IT.

C. Aptamer Selection Scheme

Affinity selections were performed separately with each candidatemixture, comparing binding between target protein beads (signal, S) and(His)₆ beads (background, B). For each sample, a 0.5 μM candidate DNAmixture was prepared in 40 μL SB1T. 1 μL (His)₆-complement oligo (1 mM)(FIG. 3) was added to the DNA, along with 10 μL of a protein competitormixture (0.1% HSA, 10 μM casein, and 10 μM prothrombin in SB1T).

Binding reactions were performed by adding 50 μL target protein-coatedbeads or (His)₆-coated beads (5 mg/mL in SB1T) to the DNA mixture andincubating 37° C. for 15 minutes with mixing. The DNA solution wasremoved and the beads were washed 5 times at 37° C. with SB1T containing0.1 mg/mL herring sperm DNA (Sigma-Aldrich). Unless indicated, allwashes were performed by re-suspending the beads in 100 μL washsolution, mixing for 30 seconds, separating the beads with a magnet, andremoving the wash solution. Bound aptamers were eluted from the beads byadding 100 μL SB1T+2 M Guanidine-HCl and incubating at 37° C. for 5minutes with mixing. The aptamer eluate was transferred to a new tubeafter magnetic separation. After the first two selection rounds, thefinal two of five target beads washes were done for 5 minutes instead of30 seconds.

Primer beads were prepared by immobilizing biotinylated reverse PCRprimer to streptavidin-coated paramagnetic beads (MyOne-Streptavidin C1(SA beads), Invitrogen). 5 mL SA beads (10 mg/mL) were washed once withNaClT (5 M NaCl, 0.01% TWEEN-20), and resuspended in 5 mL biotinylatedreverse PCR primer (5 μM in NaClT). The sample was incubated at 25° C.for 15 minutes, washed twice with 5 mL NaClT, resuspended in 12.5 mLNaClT (4 mg/mL), and stored at 4° C.

25 μL primer beads (4 mg/mL in NaClT) were added to the 100 μL aptamersolution in Guanidine Buffer and incubated at 50° C. for 15 minutes withmixing. The aptamer solution was removed, and the beads were washed 5times with SB1T. Aptamer was eluted from the beads by adding 85 μL 20 mMNaOH, and incubating at 37° C. for 1 minute with mixing. 80 μL aptamereluate was transferred to a new tube after magnetic separation,neutralized with 20 μL 80 mM HCl, and buffered with 1 μL 0.5M Tris-HCl,pH 7.5.

D. Aptamer Amplification and Purification

Selected aptamer DNA was amplified and quantified by QPCR. 48 μL DNA wasadded to 12 μL QPCR Mix (5×KOD DNA Polymerase Buffer, 25 mM MgCl₂, 10 μMforward PCR primer, 10 mM biotinylated reverse PCR primer, 5×SYBR GreenI, 0.125 U/μL KOD XL DNA Polymerase, and 1 mM each dATP, dCTP, dGTP, anddTTP) and thermal cycled in an ABI5700 QPCR instrument with thefollowing protocol: 1 cycle of 99.9° C., 15 seconds, 55° C., 10 seconds,70° C., 30 minutes; 30 cycles of 99.9° C., 15 seconds, 72° C., 1 minute.Quantification was done with the instrument software and the number ofcopies of DNA selected with target beads and (His)₆ beads were comparedto determine signal/background ratios.

Following amplification, the PCR product was captured on SA beads viathe biotinylated antisense strand. 1.25 mL SA beads (10 mg/mL) werewashed twice with 0.5 mL 20 mM NaOH, once with 0.5 mL SB1T, resuspendedin 2.5 mL 3 M NaCl, and stored at 4° C. 25 μL SA beads (4 mg/mL in 3 MNaCl) were added to 50 μL double-stranded QPCR product and incubated at25° C. for 5 minutes with mixing. The beads were washed once with SB1T,and the “sense” strand was eluted from the beads by adding 200 μL 20 mMNaOH, and incubating at 37° C. for 1 minute with mixing. The elutedstrand was discarded and the beads were washed 3 times with SB1T andonce with 16 mM NaCl.

Aptamer sense strand was prepared with the appropriate nucleotidecomposition by primer extension from the immobilized antisense strand.The beads were resuspended in 20 μL primer extension reaction mix (1×Primer Extension Buffer (120 mM Tris-HCl, pH 7.8 @ 20, 10 mM KCl, 7 mMMgSO₄, 6 mM (NH₄)₂SO₄, 0.001% BSA, and 0.01% Triton X100), 5 μM forwardPCR primer, 0.125 U/μL KOD XL DNA Polymerase, 0.5 mM each dATP, MedCTP,dGTP, and either dTTP or dUTP analog) and incubated at 68° C. for 30minutes with mixing. The beads were washed 3 times with SB1T, and theaptamer strand was eluted from the beads by adding 85 μL 20 mM NaOH, andincubating at 37° C. for 1 minute with mixing. 80 μL aptamer eluate wastransferred to a new tube after magnetic separation, neutralized with 20μL 80 mM HCl, and buffered with 5 μL 0.1 M HEPES, pH 7.5.

E. Selection Stringency and Feedback

The relative target protein concentration of the selection step waslowered each round in response to the S/B ratio as follows, where signalS and background B are defined in Section C above:

if S/B<10, [P](i+1)=[P]i

if 10≦S/B<100, [P](i+1)=[P]i/3.2

if S/B≧100, [P](i+1)=[P]i/10

where [P]=protein concentration and i=current round number.

Target protein concentration was lowered by adjusting the mass of targetprotein beads (and (His)₆ beads for background determination) added tothe selection step.

After each selection round, the convergence state of the enriched DNAmixture was determined. 5 μL double-stranded QPCR product was diluted to200 μL with 4 mM MgCl₂ containing 1×SYBR Green I. Samples were overlaidwith 75 μL silicon oil and analyzed for convergence using a C₀t analysiswhich measures the hybridization time for complex mixtures of doublestranded oligonucleotides. The sample was thermal cycled with thefollowing protocol: 3 cycles of 98° C., 1 minute, 85° C., 1 minute; 1cycle of 93° C., 1 minute, 85° C., 15 minutes. During the 15 minutes at85° C., fluorescent images were measured at 5-second intervals. Thefluorescence intensity was plotted as a function of log (time) toevaluate the diversity of the sequences.

F. Measurement of Equilibrium Binding Constant (Kd)

Equilibrium binding constants of the enriched libraries were measuredusing TALON bead partitioning. DNA was renatured by heating to 95° C.and slowly cooling to 37° C. Complexes were formed by mixing a lowconcentration of radiolabled DNA (˜1×10⁻¹¹ M) with a range ofconcentrations of target protein (1×10⁻⁷ M to 1×10⁻¹² M final) in SB1Buffer, and incubating at 37° C. A portion of each reaction wastransferred to a nylon membrane and dried to determine total counts ineach reaction. A small amount of 5 mg/mL TALON beads was added to theremainder of each reaction and mixed at 37° C. for one minute. A portionwas passed through a MultiScreen HV Plate (Millipore) under vacuum toseparate protein-bound complexes from unbound DNA and washed with 100 μLSB1 Buffer. The nylon membranes and MultiScreen HV Plates werephosphorimaged and the amount of radioactivity in each sample quantifiedusing a FUJI FLA-3000. The fraction of captured DNA was plotted as afunction of protein concentration and a non-linear curve-fittingalgorithm was used to extract equilibrium binding constants (K_(d)values) from the data. Table 1 shows the K_(d) values determined foreach enriched candidate mixture to a set of targets. NT indicates thatthe enriched library for a particular base composition did not appear tohave changed from the original candidate mixture, as determined by C₀tanalysis, and was therefore Not Tested (NT).

Table 1 shows the equilibrium binding constants (K_(d)) for enrichedpools to fifteen different protein targets and four different DNAlibraries: naturally occurring bases (dT), benzyl (BzdU), isobutyl(iBdU) or tryptophan (TrpdU). An aptamer with a K_(d) of less than1×10⁻⁸ is desirable. The use of modified bases in the SELEX processproduces a significantly higher percentage of desirable high affinityaptamers. It was observed that only 2 of the 14 aptamers produced withthe normal nucleotides have the desired slow dissociation rates. Slowoff-rate aptamers produced with the modified nucleotides were identified9 of 14, 7 of 14, and 14 of 14 for BzdUTP, iBdUTP, and TrpdUTP,respectively.

TABLE 1 Equilibrium binding constants (K_(d)) of the enriched librariesselected with different modified nucleotides, reported in units ofmolarity. Target Protein dTTP BzdUTP iBdUTP TrpdUTP 4-1BB >1.0 × 10⁻⁷5.6 × 10⁻⁹ >1.0 × 10⁻⁷   3.9 × 10⁻⁹ B7 >1.0 × 10⁻⁷ 1.1 × 10⁻⁸ NT 7.2 ×10⁻⁹ B7-2 >1.0 × 10⁻⁷ NT >1.0 × 10⁻⁷   5.7 × 10⁻⁹ CTLA-4 >1.0 × 10⁻⁷ NTNT 1.4 × 10⁻⁹ E-Selectin >1.0 × 10⁻⁷ >1.0 × 10⁻⁷   >1.0 × 10⁻⁷   1.9 ×10⁻⁹ Fractalkine NT >1.0 × 10⁻⁷   NT  5.1 × 10⁻¹¹ GA733-1   8.9 × 10⁻⁹2.8 × 10⁻⁹ 4.7 × 10⁻⁹  4.5 × 10⁻¹⁰ protein Gp130 >1.0 × 10⁻⁷ 5.9 × 10⁻⁹2.2 × 10⁻⁸ 1.2 × 10⁻⁹ HMG-1 >1.0 × 10⁻⁷ NT 2.2 × 10⁻⁸ 4.9 × 10⁻⁹ IR >1.0× 10⁻⁷ 1.9 × 10⁻⁹ 1.2 × 10⁻⁸  2.2 × 10⁻¹⁰ OPG   3.7 × 10⁻⁸ 4.6 × 10⁻⁹9.5 × 10⁻⁹  1.7 × 10⁻¹⁰ PAI-1 >1.0 × 10⁻⁷  3.7 × 10⁻¹⁰  9.1 × 10⁻¹⁰  4.3× 10⁻¹⁰ P-Cadherin >1.0 × 10⁻⁷ 3.5 × 10⁻⁹ 5.2 × 10⁻⁹ 2.7 × 10⁻⁹ sLeptinR >1.0 × 10⁻⁷ 2.3 × 10⁻⁹ NT  4.6 × 10⁻¹⁰ NT = not tested.

Example 2 Generation of PhotoAptamers Using 5′-Fixed PhotoSELEX and SlowOff-Rate Enrichment Process by Dilution

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and BzdUTP were preparedby polymerase extension of a primer annealed to a biotinylated template(FIG. 5A-B). For each template, four different forward primers wereused, each possessing a unique chromophore at the 5′ terminus (see FIG.6 for the chromophore structures). For each candidate mixture, 11 nmolforward primer (with 5′ chromophore) and 10 nmol template were combinedin 250 μL Primer Extension Buffer (120 mM Tris-HCl, pH 7.8, 10 mM KCl, 6mM (NH₄)₂SO₄, 7 mM MgSO₄, 0.1 mg/mL BSA, 0.1% Triton X-100), heated to95° C. for 5 minutes, and cooled on ice. 125 μL each primer:templatemixture was added to a 1 mL extension reaction containing PrimerExtension Buffer, 0.125 U/μL KOD XL DNA Polymerase, and 0.5 mM eachdATP, dCTP, dGTP, and BzdUTP, and incubated at 70° C. for 30 minutes.Each 1 mL reaction was split into four 250 μL aliquots and chilled onice. Double-stranded product was captured via the template strandbiotins by adding 1 mL streptavidin-coated magnetic beads(MagnaBind-Streptavidin, Pierce, 5 mg/mL in 1M NaCl+0.05% TWEEN-20) toeach 250 μL aliquot and incubating at 25° C. for 60 minutes with mixing.Beads were washed three times with 0.5 mL SB17T Buffer (40 mM HEPES, pH7.5, 125 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 0.05% TWEEN-20). Theaptamer strand was eluted from the beads with 1 mL 20 mM NaOH,neutralized with 0.25 mL 80 mM HCl, and buffered with 10 μL 1 M HEPES,pH 7.5. Candidate mixtures were concentrated with a Centricon-30 toapproximately 0.2 mL, and quantified by UV absorbance spectroscopy.

B. Preparation of Target Proteins

Untagged target proteins were biotinylated by covalent coupling ofNHS-PEO4-biotin (Pierce) to lysines residues. Proteins (300 pmol in 50μL) were exchanged into SB17T with a Sephadex G-25 microspin column.NHS-PEO4-biotin was added to 1.5 mM and the reaction was incubated at 4°C. for 16 hours. Unreacted NHS-PEO4-biotin was removed with a SephadexG-25 microspin column.

C. Aptamer Selection with Slow Off-Rate Enrichment Process andPhotocrosslinking

Selections were performed separately with each candidate mixture,comparing binding between samples with target protein (signal S) andsamples without target protein (background B). The first three roundswere performed with selection for affinity (no photocrosslinking); thesecond and third included slow off-rate enrichment process. Rounds fourthrough eight included both slow off-rate enrichment process andphotocrosslinking.

For each sample, a 90 μL DNA mixture was prepared in SB17T with 10-20pmoles candidate mixture (100 pmoles in the first round) and 100 pmolesreverse primer. Samples were heated to 95° C. for 3 minutes and cooledto 37° C. at a rate of 0.1 C/second. Samples were combined with 10 μLprotein competitor mixture (0.1% HSA, 10 μM casein, and 10 μMprothrombin in SB17T), added to 0.5 mg SA beads (pre-washed twice with20 mM NaOH and once with SB17T), and incubated at 37° C. for 5 minuteswith mixing. Beads were removed by magnetic separation.

Binding reactions were performed by adding 10 μL target protein (0.5 mMin SB17T) or SB17T to 40 μL DNA mixture and incubating at 37° C. for 30minutes.

When slow off-rate enrichment process was employed, samples were diluted20× by adding 950 μL SB17T (preheated to 37° C.), and incubated at 37°C. for 30 minutes prior to capturing complexes.

Complexes were captured on SA beads via protein biotins by adding 0.25mg MyOne-SA beads (Invitrogen) and incubating at 37° C. for 15 minuteswith mixing. Free DNA was removed by washing the beads five times withSB17T. Unless indicated, all washes were performed by resuspending thebeads in 100 μL wash solution, mixing for 30 seconds at 25° C.,separating the beads with a magnet, and removing the wash solution. Theaptamer strand was eluted from the beads by adding 85 μL 20 mM NaOH, andincubating at 37° C. for 1 minute with mixing. 80 μL aptamer eluate wastransferred to a new tube after magnetic separation, neutralized with 20μL 80 mM HCl, and buffered with 1 μL 0.5 M Tris-HCl, pH 7.5.

When photo-selection was employed, the 50 μL binding reactions, (or 1 mLbinding reactions after optional slow off-rate enrichment process bydilution) were irradiated from above with a high-pressure mercury lamp(Optical Associates, Inc. model 0131-0003-01, 500 W, with 310 nm mirrorset). Candidate mixtures possessing a BrdU chromophore were irradiatedfor 37 seconds, those possessing an ANA chromophore were irradiated for60 seconds, and those possessing an AQ or psoralen chromophore wereirradiated for 10 minutes. An additional filter (5 mm plate glass) wasused for the ANA, AQ and psoralen chromophores to eliminate unnecessary,but potentially damaging wavelengths below 320 nm. Complexes werecaptured as above, and non-crosslinked DNA was removed by washing thebeads once with 4 M guanidine-HCl+0.05% TWEEN-20 at 50° C. for 10minutes, once with 20 mM NaOH at 25° C. for 2 minutes, twice with SB17T,and once with 16 mM NaCl. Crosslinked DNA was not removed from the beadsurface for the amplification steps.

D. Aptamer Amplification and Purification

Selected aptamer DNA was amplified and quantified by QPCR. 48 μL DNA wasadded to 12 μL QPCR Mix (5×KOD DNA Polymerase Buffer, 25 mM MgCl₂, 10 μMforward PCR primer, 10 μM biotinylated reverse PCR primer, 5×SYBR GreenI, 0.125 U/μL KOD XL DNA Polymerase, and 1 mM each dATP, dCTP, dGTP, anddTTP) and thermal cycled in an a Bio-Rad MyIQ QPCR instrument with thefollowing protocol: 1 cycle of 99.9° C., 15 sec, 55° C., 10 sec, 68° C.,30 min, 30 cycles of 99.9° C., 15 seconds, 72° C., 1 minute.Quantification was done with the instrument software and the number ofcopies of DNA selected with and without target protein were compared todetermine signal/background ratios.

When photo-selection was employed, a cDNA copy of the selected DNA wasprepared by primer extension on the bead surface. Washed beads wereresuspended in 20 μL cDNA extension mix (Primer Extension Buffercontaining 5 mM reverse PCR primer, 0.5 mM each dATP, dCTP, dGTP, anddTTP, and 0.125 U/μL KOD XL DNA Polymerase) and incubated at 68° C. for30 minutes with mixing. The beads were washed 3 times with SB17T, andthe aptamer strand was eluted by from the beads by adding 85 μL 20 mMNaOH, and incubating at 37° C. for 1 minute with mixing. 80 μL aptamereluate was transferred to a new tube after magnetic separation,neutralized with 20 μL 80 mM HCl, and buffered with 1 μL 0.5 M Tris-HCl,pH 7.5. The cDNA was amplified and quantified by QPCR as above for the30 cycles of 99.9° C., 15 seconds, 72° C., 1 minute.

Following amplification, the PCR product was captured on SA beads viathe biotinylated antisense strand. 1.25 mL SA beads (10 mg/mL) werewashed twice with 0.5 mL 20 mM NaOH, once with 0.5 mL SB17T, resuspendedin 1.25 mL 3 M NaCl+0.05% Tween, and stored at 4° C. 25 μL SA beads (10mg/mL in 3 M NaClT) were added to 50 μL double-stranded QPCR product andincubated at 25° C. for 5 minutes with mixing. The beads were washedonce with SB17T, and the “sense” strand was eluted from the beads byadding 200 μL 20 mM NaOH, and incubating at 37° C. for 1 minute withmixing. The eluted strand was discarded and the beads were washed 3times with SB17T and once with 16 mM NaCl.

Aptamer sense strand was prepared with the appropriate chromophore byprimer extension from the immobilized antisense strand. The beads wereresuspended in 20 μL primer extension reaction mixture (1× PrimerExtension Buffer, 1.5 mM MgCl₂, 5 μM forward primer with appropriate 5′chromophore, 0.5 mM each dATP, dCTP, dGTP, and BzdUTP, and 0.125 U/μLKOD XL DNA Polymerase) and incubated at 68° C. for 30 minutes withmixing. The beads were washed 3 times with SB17T, and the aptamer strandwas eluted from the beads by adding 85 μL 20 mM NaOH, and incubating at37° C. for 1 minute with mixing. 80 μL aptamer eluate was transferred toa new tube after magnetic separation, neutralized with 20 μL 80 mM HCl,and buffered with 5 μL 0.1 M HEPES, pH 7.5.

E. Selection Stringency and Feedback

Target protein was adjusted at each round as described in Example 1.After each round of selection, the convergence state of the enrichedpool was determined as described in Example 1.

F. Equilibrium Binding Constants of Enriched Libraries

The binding affinity was determined as described in Example 1 above, butwith SA capture beads. The following table, Table 2, summarizes theequilibrium binding constants (K_(d)) obtained using the photoSELEXprotocol with slow off-rate enrichment process.

TABLE 2 Equilibrium binding constants (K_(d)) of the enriched librariesselected with different chromophores, reported in units of molarity.Target Protein BrdU AQ ANA Psor β-catenin 2.7 × 10⁻⁸ 3.6 × 10⁻⁹  1.1 ×10⁻⁹  1.6 × 10⁻⁹ bFGF 3.1 × 10⁻⁸ 5.7 × 10⁻¹⁰ 7.1 × 10⁻¹⁰  5.1 × 10⁻¹⁰CMP-SAS x 6.2 × 10⁻⁹  7.3 × 10⁻⁹  4.9 × 10⁻⁸ endostatin 1.3 × 10⁻⁹ 8.7 ×10⁻¹⁰ 8.8 × 10⁻¹⁰ 1.3 × 10⁻⁹ IL-6 1.0 × 10⁻⁹ 5.4 × 10⁻¹⁰ 4.0 × 10⁻¹⁰ xmyeloper-  6.0 × 10⁻¹⁰ 2.8 × 10⁻¹⁰ 5.0 × 10⁻¹⁰  1.5 × 10⁻¹⁰ oxidaseSDF-1β  8.1 × 10⁻¹⁰ 5.7 × 10⁻¹⁰ 3.8 × 10⁻¹⁰ x TIMP-1 5.2 × 10⁻⁹ 7.3 ×10⁻⁹  8.9 × 10⁻⁹  x VEGF  7.2 × 10⁻¹⁰ 4.2 × 10⁻⁹  5.5 × 10⁻¹⁰ x vWF 2.6× 10⁻⁸ 8.8 × 10⁻⁹  8.1 × 10⁻⁹  x Measurements were not made on librariesthat failed to converge (indicated with an x).

G. Crosslink Activity Assay

The crosslink yield of enriched libraries was determined by measuringthe percent of DNA crosslinked to protein under conditions of saturatingprotein and light. Radiolabeled DNA (50 pM) was mixed with reverseprimer (16 nM) in SB17T, heated to 95° C. for 3 minutes, and cooled to37° C. at 0.1° C./second. Target protein was added to the DNA mix to afinal concentration of 10 nM and incubated at 37° C. for 30 minutes.Control samples with no protein were simultaneously prepared. Sampleswere crosslinked with the chromophore-specific conditions describedabove, but with a saturating dose (6 minutes for BrdU, 10 minutes forANA, and 30 minutes for AQ and Psor). Samples were analyzed bydenaturing PAGE, FIG. 7, and quantified and the results are tabulated inTable 3.

TABLE 3 Crosslink yields of the enriched libraries selected withdifferent chromophores, reported in units of percent of total DNAcrosslinked to protein. Target Protein BrdU AQ ANA Psor β-catenin 15 9 81 bFGF 4 9 15 4 CMP-SAS x 3 5 2 Endostatin 2 1 18 3 IL-6 0 5 9Myeloperoxidase 4 13 9 8 SDF-1β 8 10 17 x TIMP-1 1 4 2 x VEGF 1 1 4 xvWF 2 2 7 x Measurements were not made on libraries that failed toconverge (indicated with an x).

Example 3 Generation of Slow off-Rate Aptamers Using a Slow off-RateEnrichment Process with a Competitor

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and BzdUTP were preparedby polymerase extension of a primer annealed to a biotinylated templatefor 94 protein targets. 55 nmol forward primer (with 5′ ANA chromophore)and 55 nmol template were combined in 0.5 mL Primer Extension Buffer(120 mM Tris-HCl, pH 7.8, 10 mM KCl, 6 mM (NH₄)₂SO₄, 7 mM MgSO₄, 0.1mg/mL BSA, 0.1% Triton X-100), heated to 95° C. for 5 minutes, 70° C.for 5 minutes, 48° C. for 5 minutes, and cooled on ice. Theprimer:template mixture was added to a 5.5 mL extension reactioncontaining Primer Extension Buffer, 0.125 U/μL KOD XL DNA Polymerase,and 0.5 mM each dATP, dCTP, dGTP, and BzdUTP, and incubated at 70° C.for 60 minutes. After completion of the extension reaction, the solutionwas chilled on ice. Double-stranded product was captured via thetemplate strand biotins by adding 25 mL streptavidin-coated magneticbeads (MagnaBind-Streptavidin, Pierce, 5 mg/mL in 1 M NaCl+0.05%TWEEN-20) to the primer extension product and incubating 25° C. for 15minutes with rotating. Beads were washed three times with 40 mL SB17TBuffer (40 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 1 mMEDTA, 0.05% TWEEN-20). The aptamer strand was eluted from the beads with35.2 mL 20 mM NaOH for 5 minutes with shaking. The eluted strand wasneutralized with 8.8 mL 80 mM HCl, and buffered with 400 μL 1 M HEPES,pH 7.3. Candidate mixtures were concentrated with a Centricon-30 toapproximately 0.7 mL, and quantified by UV absorbance spectroscopy.

B. Preparation of Target Proteins

Untagged target proteins were biotinylated as described in Example 2.

C. Aptamer Selection with Slow off-rate Enrichment Process andPhotocrosslinking

Selections were performed separately as described in Example 2, with theaddition of 10 mM dextran sulfate as a competitor for aptamer rebindingduring the slow off-rate enrichment process in rounds six through nine.

The slow off-rate enrichment process was employed in three differentways. In rounds two and three, samples were diluted 20× by adding 950 μLSB17T (preheated to 37° C.), and incubated at 37° C. for 30 minutesprior to capturing complexes. In rounds four and five, samples werediluted 20× by adding 950 μL SB17T (preheated to 37° C.), and incubatedat 37° C. for 30 minutes prior to crosslinking. In rounds six and seven,samples were diluted 20× by adding 950 μL SB17T (preheated to 37° C.).50 μL of each diluted sample was diluted again by transferring to 950 μLSB17T+10 mM 5000K dextran sulfate (preheated to 37° C.) to give anoverall 400× dilution, and incubated at 37° C. for 60 minutes prior tocrosslinking. In rounds eight and nine, samples were diluted 20× byadding 950 μL SB17T (preheated to 37° C.), and 50 μL of each sample wasdiluted again by transferring to 950 μL SB17T (preheated to 37° C.) togive 400× dilution. Finally, 50 μL of each 400× diluted sample wasdiluted again by transferring to 950 μL SB17T+10 mM 5000K dextransulfate (preheated to 37° C.) to give an overall 8000× dilution, andincubated at 37° C. for 60 minutes prior to crosslinking. Complexes werecaptured and washed as described in Example 2. When photo-crosslinkingwas employed, the 1 mL binding reactions after the slow off-rateenrichment process were irradiated from above with an array of 470 nmLEDs for 60 seconds prior to complex capture as in Example 2.

D. Aptamer Amplification and Purification

Amplification and purification were performed as in Example 2.

E. Selection Stringency and Feedback

Target protein was adjusted at each round as described in Example 1,except in rounds six and eight. In order to maximize signal after theselarge dilutions, the target protein was increased to 100 nM for roundssix and eight. After each round of selection, the convergence state ofthe enriched pool was determined as described in Example 1.

F. Dissociation Rate Constant Determination Protocol.

The rate constant for aptamer:protein complex dissociation (koff) wasdetermined for each aptamer by measuring the fraction of pre-formedaptamer:protein complexes that remain bound after dilution as a functionof time. Radiolabeled aptamer (50 μM) was equilibrated in SB17T-0.002(SB17T with TWEEN-20 reduced to 0.002%) at 37° C. with protein at aconcentration 10× greater than the measured K_(d) value. Samples werediluted 100× with SB17T-0.002 at 37° C. and aliquots were removed atvarious time points and partitioned to separate free aptamer fromprotein:aptamer complexes. Partitioning was accomplished by addingZORBAX resin (Agilent) to the sample, capturing complexes on the resin,passing the sample through a DuraPore membrane under vacuum, and washingthe resin with SB17T-0.002. For proteins not efficiently captured withZORBAX resin, the assay was performed with biotinylated protein in SB17Tand partitioning was accomplished by capturing complexes with SA beads.The amount of complex remaining at each time point was determined byquantifying the radiolabeled aptamer on the resin with a FUJI FLA-3000phosphorimager. The fraction of complex was plotted as a function oftime and the dissociation rate constant (koff) and dissociationhalf-life value (t_(1/2)) was determined by fitting the data to ananalytic expression for bimolecular dissociation kinetics usingnon-linear regression.

G. Kinetic Properties of some Aptamers

The following table, Table 4, summarizes the dissociation half-lifevalues (t_(1/2)) obtained for aptamers selected against 10 targets usingthis protocol.

TABLE 4 Dissociation half-life values (t_(1/2)) of aptamers using thecompetitor slow off-rate enrichment step protocol. Target Proteint_(1/2) (min) bFGF R 66 C3 164 catalase 58 FGF-17 91 group IBphospholipase A2 40 HB-EGF 49 HCC-4 143 IL-6 sRα 114 SAP 186 uPA 85

Example 4 The Slow Off-Rate Enrichment Process Increases theDissociation Half-Life of Selected Aptamers

Dissociation half-life values (t_(1/2)) were measured and plotted for 65aptamers that were selected by either the affinity SELEX methoddescribed in Example 1 or photo SELEX methods described in U.S. Pat. No.6,458,539, entitled “Photoselection of Nucleic Acid Ligands” without aslow off-rate enrichment process (FIG. 4A). t_(1/2) values were alsomeasured and plotted for 72 aptamers that were selected by the slowoff-rate enrichment process described in Example 2 with a slow off-rateenrichment process by dilution or dilution with competitor (FIG. 4B).The average t_(1/2) value for aptamers using the modified nucleotides5-benzyl-dUTP (BzdUTP), 5-isobutyl-dUTP (iBdUTP), or 5-tryptamino-dUTPselected in the absence of a slow off-rate enrichment process was 20minutes with some aptamers having a t_(1/2) value of up to one hour.This is substantially longer than what has been previously describedwith natural bases or other modified nucleotides. The average foraptamers selected with a slow off-rate enrichment process was over 85minutes, with some aptamers having a t_(1/2) value in excess of fourhours.

Example 5 Generation of Aptamers from a NpdUTP Random Library

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and NpdUTP were preparedas described in Example 3 but without the 5′-ANA photoreactive group.

B. Immobilization of Target Proteins

Target proteins contained a (His)₆ tag and were captured with Talonbeads as described in Example 1.

C. Aptamer Selection with Slow off-rate Enrichment Process

Aptamer selection was performed as described in Example 3, but withoutphotocrosslinking.

D. Aptamer Amplification and Purification

Amplification and purification were performed as described in Example 3.

E. Selection Stringency and Feedback

Selection stringency and feedback were performed as described in Example3.

F. Aptamer Properties

The equilibrium binding constant (K_(d)) of four aptamers from thisselection are listed in Table 5.

TABLE 5 Equilibrium binding constants (Kd) of NpdUTP aptamers TargetProtein K_(d)(M) bFGF 1.1. × 10⁻⁹  Endostatin 2.0. × 10⁻¹⁰ TIMP-3 1.5. ×10⁻¹⁰ VEGF 7.2. × 10⁻¹⁰

Example 6 Generation of Slow Off-Rate Aptamers for a Peptide TargetUsing a Slow off-rate Enrichment Process with a Competitor

A. Preparation of Candidate Mixtures

Candidate mixtures containing dATP, dCTP, dGTP, and BzdUTP were preparedby polymerase extension of a primer with a 5′ ANA chromophore andpurified as described in Example 3.

B. Aptamer Selection with Slow off-rate Enrichment Process andPhotocrosslinking

Aptamer selection was performed as described in Example 3 with the 29amino acid biotinylated target peptide SMAP29 (Sheep MyeloidAntibacterial Peptide MAP-29, Anaspec).

C. Aptamer Amplification and Purification

Amplification and purification were performed as described in Example 3.

D. Selection Stringency and Feedback

Selection stringency and feedback were performed as described in Example3.

E. Aptamer Properties

The equilibrium binding constant (K_(d)) of an aptamer from thisselection was 1.2×10⁻⁸ M (measured according to the protocol describedin Example 1). The dissociation half-life (t_(1/2)) of this aptamer was69 minutes (measured according to the protocol described in Example 3).Results are shown in FIG. 13A and FIG. 13B.

Example 7 Protein Measurements in Test Samples Were Enabled by Aptamerswith Slow Off-Rates

A. Preparation of Aptamer/Primer Mixtures and Test Samples

Aptamers with a biotin Cy3 detection label (4 nM each) were mixed with a3× excess of capture probe (oligonucleotide complementary to the 3′fixed region of the aptamer containing a biotin tag and photocleavableelement) in 1×SB17T and heated at 95° C. for 4 minutes, then 37° C. for13 minutes, and diluted 1:4 in 1×SB17T. 55 μL of aptamer/primer mix wasadded to a microtiter plate (Hybaid # AB-0407) and sealed with foil.Test samples were prepared in a microtiter plate by mixing knownconcentrations of protein analytes in SB17T and diluting serially withSB17T.

B. Sample Equilibration

55 μL of aptamer/primer mix was added to 55 μL of test sample andincubated at 37° C. for 15 minutes in a foil-sealed microtiter plate.The final concentration of each aptamer in the equilibration mixture was0.5 nM. After equilibration, all subsequent steps of this method wereperformed at room temperature unless otherwise noted.

C. Aptamer Capture and Free Protein Removal

A DuraPore filtration plate (Millipore HV cat# MAHVN4550) was washedonce with 100 μL 1×SB17T by vacuum filtration, 133.3 μL 7.5%Streptavidin-agarose resin (Pierce) was added to each well and washedtwice with 200 μL 1×SB17T. 100 μL of equilibrated samples wastransferred to the Durapore plate containing the Streptavidin-agaroseresin and incubated on a thermomixer (Eppendorf) at 800 rpm for 5minutes. The resin was washed once with 200 μL 1×SB17T+100 mM biotin andonce with 200 μL 1×SB17T.

D. Protein Tagging with Biotin

100 μL of 1.2 mM NHS-PEO4-biotin in SB17T, prepared immediately beforeuse, was added to the resin with captured aptamer and aptamer:proteincomplexes and incubated on a thermomixer at 800 rpm for 20 minutes. Theresin was washed five times with 200 μL 1×SB17T by vacuum filtration.

E. Slow Off-Rate Enrichment Process & Photocleavage

The drip director was removed from underside of the DuraPore plate andthe plate was placed over a 1 mL microtiter collection plate. The resinwas washed once with 200 μL 1×SB17T by centrifugation at 1000×g for 30sec. 80 μL of 1×SB17T+10 mM dextran sulfate was added to the resin andirradiated with a BlackRay Mercury Lamp on a thermomixer at 800 rpm for10 minutes. The DuraPore plate was transferred to a new 1 mL deepwellplate and centrifuged at 1000×g for 30 seconds to collect thephotocleaved aptamer and protein:aptamer complexes.

F. Protein Capture and Free Aptamer Removal

50 μL of MyOne-streptavidin C1 paramagnetic beads (Invitrogen) (10 mg/mLin 1×SB17T) was added to a microtiter plate. The beads were separatedwith a magnet for 60 seconds and the supernatant was removed. 225 μL ofphotocleavage mixture was added to the beads and mixed for 5 minutes.The beads were washed four times with 200 μL 1×SB17T by separating themagnetic beads and replacing the wash buffer. The final wash buffer wasremoved.

G. Aptamer Elution

100 μL Sodium Phosphate Elution Buffer (10 mM Na₂HPO₄, pH 11) was addedto the beads and mixed for 5 minutes. 90 μL of eluate was transferred toa microtiter plate and neutralized with 10 μL Sodium PhosphateNeutralization Buffer (10 mM NaH₂PO₄, pH 5).

H. Aptamer Hybridization to Microarrays

DNA arrays were prepared with oligonucleotide capture probes comprisedof the complementary sequence of the variable region of each aptamerimmobilized on a custom microscope slide support. Multiple arrays(subarrays) exist on each slide, and subarrays were physically separatedby affixing a gasket (Grace) for sample application. Arrays werepretreated with 100 μL Blocking Buffer and incubated for 15 minutes at65° C. on a thermomixer. 30 μL of high salt Hybridization Buffer wasadded to 90 μL of neutralized aptamer eluate in a microtiter plate,incubated at 95° C. for 5 minutes in a thermalcycler, and cooled to 65°C. at 0.1° C./second. Blocking Buffer was removed from the arrays and110 μL of aptamer sample was added to the arrays and incubate in a humidchamber at 65° C. for 20 hours.

I. Array Washing

Aptamer sample was removed from the arrays, and the arrays were washedonce with 200 μL of sodium phosphate Tween-20 wash buffer at 65° C.,with the gasket in place, and three times with 25 mL sodium phosphate,Tween-20 wash buffer at 65° C. in a pap jar with the gasket removed.Arrays were dried with a nitrogen gun.

J. Quantitate Signal On Arrays

Array slides were scanned on a TECAN LS300 Reloaded in an appropriatechannel for Cy3 detection and Cy3 signal on each array feature isquantified.

Results:

Apatmers specific to three different targets (bFGF, VEGF, andMyeloperoxidase) were produced using traditional SELEX methods andmaterials. A second set of aptamers specific to the same set of targetswere made using 5-position modified nucleotides and selected for veryslow off-rates for their respective targets. Aptamers made in thetraditional process had measured off-rates on the order of less than 5minutes. Aptamers made with the modified nucleotides and using slowoff-rate enrichment process during selection had off-rates of greaterthan 20 minutes. Two sets of aptamers were made for each target by thetwo different methods for a total of 4 different aptamer populations foreach target. The ability of these aptamer populations to measure analyteconcentrations in test samples was evaluated as described above over arange of target concentrations. Relative signal from the DNA chipdetection was plotted against the input target concentration. See FIG.12A to 12C. The response curve of the traditional aptamers is very flatand the sensitivity of the detection is fairly low. The sensitivity ofdetection of the respective targets with the slow off-rate aptamers isexcellent. The data supports the need to use the slow off-rate aptamersfor maximum analytic performance.

Example 8 Generation of High Affinity BzdU Aptamers to Human Thrombin

A. Preparation of Candidate Mixture

A candidate mixture containing dATP, dCTP, dGTP, and BzdUTP was preparedby polymerase extension of a primer with a 5′ ANA chromophore andpurified as described in Example 3.

B. Preparation of Target Protein

Human thrombin was tagged with biotin as describe in Example 2.

C. Aptamer Selection with Slow Off-Rate Enrichment and Photocrosslinking

Aptamer selection was performed as described in Example 3 withbiotinylated human thrombin as the target.

D. Aptamer Amplification and Purification

Amplification and purification were performed as described in Example 3.

E. Selection Stringency and Feedback

Selection stringency and feedback were performed as described in Example3.

F. Aptamer Properties

The equilibrium binding constant (K_(d)) of aptamer 2336-17 from thisselection with a modified BzdU was 4.4×10⁻¹¹ M (measured according tothe protocol described in Example 1) as demonstrated in FIG. 15. In theart, single-stranded DNA aptamers to human thrombin were selected from alibrary comprised of natural dA, dC, dG, and dT nucleotides (Bock, etal., “Selection of Single-Stranded DNA Molecules that Bind and InhibitHuman Thrombin,” Nature (1992) 355:564-566). The binding affinities ofthe aptamers had K_(d) values ranging from 2.5×10⁻⁸ M to 2.0×10⁻⁷ M.Using a similar protocol with a library comprised of natural dA, dC, dG,and modified 5-(1-pentynyl)-dUTP, aptamers were selected with K_(d)values ranging from 4×10⁻⁷ M to 1×10⁻⁶ M (Latham, et al., “TheApplication of a Modified Nucleotide in Aptamer Selection: NovelThrombin Aptamers Containing 5-(1-Pentynyl)-2′-Deoxyuridine,” NucleicAcid Research (1994) 22(14): 2817-2822).

A number of patents, patent application publications, and scientificpublications are cited throughout and/or listed at the end of thedescription. Each of these is incorporated herein by reference in theirentirety. Likewise, all publications mentioned in an incorporatedpublication are incorporated by reference in their entirety.

Examples in cited publications and limitations related therewith areintended to be illustrative and not exclusive. Other limitations of thecited publications will become apparent to those of skill in the artupon a reading of the specification and a study of the drawings.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively.

1. A histology or cytology reagent comprising a slow off-rate aptamer,wherein said aptamer has a rate of dissociation (t_(1/2)) greater thanor equal to about 30 minutes.
 2. The reagent of claim 1 wherein saidaptamer has rate of dissociation (t_(1/2)) between about 30 minutes andabout 240 minutes.
 3. The reagent of claim 1 wherein said aptamer has arate of dissociation (t1/2) selected from the group consisting of a time≧about 30 minutes, ≧about 60 minutes, ≧about 90 minutes, ≧about 120minutes, ≧about 150 minutes, ≧about 180 minutes, ≧about 210 minutes and≧about 240 minutes.
 4. An aptamer selected and produced to a targetindicative of a specific disease state wherein the target is containedin a histological or cytological specimen and further wherein saidaptamer is a slow off-rate aptamer, wherein said aptamer has a rate ofdissociation (t_(1/2)) greater than or equal to about 30 minutes.
 5. Theaptamer of claim 4 wherein said aptamer has rate of dissociation(t_(1/2)) between about 30 minutes and about 240 minutes.
 6. The aptamerof claim 4 wherein said aptamer has a rate of dissociation (t1/2)selected from the group consisting of a time ≧about 30 minutes, ≧about60 minutes, ≧about 90 minutes, ≧about 120 minutes, ≧about 150 minutes,≧about 180 minutes, ≧about 210 minutes and ≧about 240 minutes.
 7. Theaptamer of claim 4, wherein the histological or cytological specimen isfixed prior to selection of the target specific aptamer.
 8. A method fordiagnosis of a specific disease state wherein multiple tissue or cellsections are used said method comprising: a) obtaining a tissue or cellsample and sectioning said sample into multiple sections; b) reactingone of said sections with one or more slow off-rate aptamers in abuffered solution to targets contained within said tissue or cellsection; c) comparing the results obtained in step b) with a negativecontrol prepared by reacting a second section of said cell/tissue sampleprepared by treatment of said second section with said bufferedsolution; d) comparing the results obtained in step b) with a thirdsection prepared by staining said third section for the morphologicalanalysis appropriate to the tissue or cell type and disease state; ande) diagnosing said disease state.
 9. A method for identifying a slowoff-rate aptamer to a component of a fixed biological tissue, whereinthe biological tissue is fixed by a method used for histology orcytology, comprising: a) preparing a candidate mixture of nucleic acids;b) contacting said candidate mixture of nucleic acids with said fixedbiological tissue, wherein nucleic acids having an increased affinity tothe fixed biological tissue relative to the candidate mixture may bepartitioned from the remainder of the candidate mixture and wherein saidnucleic acids have a specific affinity to a protein component of saidfixed biological tissue; c) exposing the nucleic acid-target moleculecomplexes to a slow off-rate enrichment process; d) partitioning thenucleic acid-target molecule complexes from the candidate mixture; e)dissociating the nucleic acid-target molecule complexes to generate freenucleic acids; f) amplifying the free nucleic acids to yield a mixtureof nucleic acids enriched in sequences that are capable of binding tothe target molecule with increased affinity; g) repeating b) through f)as necessary; and h) identifying at least one aptamer to the targetmolecule of said fixed biological tissue.
 10. A method for identifyingan aptamer comprising at least one base-modified pyrimidines, the methodcomprising: a) preparing a candidate mixture of nucleic acids, whereineach of the nucleic acids comprises at least one base-modifiedpyrimidine independently selected from the group consisting of the basemodified pyrimidines shown in FIG. 14; b) contacting the candidatemixture with a histological or cytological specimen, wherein nucleicacids having an increased affinity to the target molecule of saidspecimen relative to other nucleic acids in the candidate mixture bindthe target molecule, forming nucleic acid-target molecule complexes; c)partitioning the nucleic acid-target molecule complexes from thecandidate mixture; d) dissociating the nucleic acid-target moleculecomplexes to generate free nucleic acids; e) amplifying the free nucleicacids to yield a mixture of nucleic acids enriched in sequences that arecapable of binding to the target molecule with increased affinity,whereby an aptamer to the target molecule may be identified; f)repeating b) through e) as necessary; and g) identifying at least oneaptamer to the target molecule of the histological or cytologicalspecimen, wherein the aptamer comprises at least one base-modifiedpyrimidine.
 11. A method for detecting one or more targets of interestin a histological or cytological specimen comprising: a) obtaining atissue or cell specimen; b) preparing the tissue or cell specimen byfixing a section of the specimen; c) dehydrating the fixed tissue orcell section; d) clearing the dehydrated tissue or cell section; e)immobilizing the cleared tissue or cell section to a microscope slide;f) washing the immobilized tissue or cell section; g) blocking thewashed tissue or cell section; h) reacting the blocked tissue or cellsection obtained in step f) with slow off-rate aptamers to said one ormore targets of interest, and i) detecting said slow off-rate aptamersas an indication of the presence or absence of said one or more targetsof interest.
 12. The method of claim 11 wherein an antigen retrievalprocess is included between steps e) and f).
 13. The method of claim 11wherein a permeabilization step is included between steps e) and f). 14.The method of claim 11 wherein additional steps comprising i) stainingwith one or more morphological stains occurs between steps e) and f),and further ii) a microscopic evaluation is completed for morphologicalstructures prior to step f), and further iii) a destaining step iscompleted prior to step f).
 15. The method of claim 14, wherein apermeabilization step is included between steps e) and f).
 16. Themethod of claim 15, wherein a rehydrating step is added between steps g)and h).
 17. The method of claim 15, wherein a kinetic challenge isintroduced between steps h) and i).
 18. The method of claim 17, whereinsaid kinetic challenge is selected from the group consisting of adding acompetitor, diluting the aptamers, or a combination of dilution andcompetitor addition.